Apo-2 receptor agonist antibodies

ABSTRACT

Novel polypeptides, designated Apo-2, which are capable of modulating apoptosis are provided. Compositions including Apo-2 chimeras, nucleic acid encoding Apo-2, and antibodies to Apo-2 are also provided.

RELATED APPLICATIONS

This application is a continuation application of Ser. No. 10/052,798filed Nov. 2, 2001 now U.S. Pat. No. 7,314,619, which is a divisionalapplication of Ser. No. 09/079,029 filed May 14, 1998, now issued asU.S. Pat. No. 6,342,369, claiming priority under Section 119(e) toprovisional application No. 60/046,615 filed May 15, 1997 andprovisional application No. 60/074,119 filed Feb. 9, 1998, the contentsof all of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to the identification,isolation, and recombinant production of novel polypeptides, designatedherein as Apo-2, and to anti-Apo-2 antibodies.

BACKGROUND OF THE INVENTION

Apoptosis or “Programmed Cell Death”

Control of cell numbers in mammals is believed to be determined, inpart, by a balance between cell proliferation and cell death. One formof cell death, sometimes referred to as necrotic cell death, istypically characterized as a pathologic form of cell death resultingfrom some trauma or cellular injury. In contrast, there is another,“physiologic” form of cell death which usually proceeds in an orderly orcontrolled manner. This orderly or controlled form of cell death isoften referred to as “apoptosis” [see, e.g., Barr et al.,Bio/Technology, 12:487-493 (1994); Steller et al., Science,267:1445-1449 (1995)]. Apoptotic cell death naturally occurs in manyphysiological processes, including embryonic development and clonalselection in the immune system [Itoh et al., Cell, 66:233-243 (1991)].Decreased levels of apoptotic cell death have been associated with avariety of pathological conditions, including cancer, lupus, and herpesvirus infection [Thompson, Science, 267:1456-1462 (1995)]. Increasedlevels of apoptotic cell death may be associated with a variety of otherpathological conditions, including AIDS, Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis,retinitis pigmentosa, cerebellar degeneration, aplastic anemia,myocardial infarction, stroke, reperfusion injury, and toxin-inducedliver disease [see, Thompson, supra].

Apoptotic cell death is typically accompanied by one or morecharacteristic morphological and biochemical changes in cells, such ascondensation of cytoplasm, loss of plasma membrane microvilli,segmentation of the nucleus, degradation of chromosomal DNA or loss ofmitochondrial function. A variety of extrinsic and intrinsic signals arebelieved to trigger or induce such morphological and biochemicalcellular changes [Raff, Nature, 356:397-400 (1992); Steller, supra;Sachs et al., Blood, 82:15 (1993)]. For instance, they can be triggeredby hormonal stimuli, such as glucocorticoid hormones for immaturethymocytes, as well as withdrawal of certain growth factors[Watanabe-Fukunaga et al., Nature, 356:314-317 (1992)]. Also, someidentified oncogenes such as myc, rel, and E1A, and tumor suppressors,like p53, have been reported to have a role in inducing apoptosis.Certain chemotherapy drugs and some forms of radiation have likewisebeen observed to have apoptosis-inducing activity [Thompson, supra].

TNF Family of Cytokines

Various molecules, such as tumor necrosis factor-α (“TNF-α”), tumornecrosis factor-β (“TNF-β” or “lymphotoxin”), CD30 ligand, CD27 ligand,CD40 ligand, OX-40 ligand, 4-1BB ligand, Apo-1 ligand (also referred toas Fas ligand or CD95 ligand), and Apo-2 ligand (also referred to asTRAIL) have been identified as members of the tumor necrosis factor(“TNF”) family of cytokines [See, e.g., Gruss and Dower, Blood,85:3378-3404 (1995); Wiley et al., Immunity, 3:673-682 (1995); Pitti etal., J. Biol. Chem., 271:12687-12690 (1996); WO 97/01633 published Jan.16, 1997]. Among these molecules, TNF-α, TNF-β, CD30 ligand, 4-1BBligand, Apo-1 ligand, and Apo-2 ligand (TRAIL) have been reported to beinvolved in apoptotic cell death. Both TNF-α and TNF-β have beenreported to induce apoptotic death in susceptible tumor cells [Schmid etal., Proc. Natl. Acad. Sci., 83:1881 (1986); Dealtry et al., Eur. J.Immunol., 17:689 (1987)]. Zheng et al. have reported that TNF-α isinvolved in post-stimulation apoptosis of CD8-positive T cells [Zheng etal., Nature, 377:348-351 (1995)]. Other investigators have reported thatCD30 ligand may be involved in deletion of self-reactive T cells in thethymus [Amakawa et al., Cold Spring Harbor Laboratory Symposium onProgrammed Cell Death, Abstr. No. 10, (1995)].

Mutations in the mouse Fas/Apo-1 receptor or ligand genes (called lprand gld, respectively) have been associated with some autoimmunedisorders, indicating that Apo-1 ligand may play a role in regulatingthe clonal deletion of self-reactive lymphocytes in the periphery[Krammer et al., Curr. Op. Immunol., 6:279-289 (1994); Nagata et al.,Science, 267:1449-1456 (1995)]. Apo-1 ligand is also reported to inducepost-stimulation apoptosis in CD4-positive T lymphocytes and in Blymphocytes, and may be involved in the elimination of activatedlymphocytes when their function is no longer needed [Krammer et al.,supra; Nagata et al., supra]. Agonist mouse monoclonal antibodiesspecifically binding to the Apo-1 receptor have been reported to exhibitcell killing activity that is comparable to or similar to that of TNF-α[Yonehara et al., J. Exp. Med., 169:1747-1756 (1989)].

TNF Family of Receptors

Induction of various cellular responses-mediated by such TNF familycytokines is believed to be initiated by their binding to specific cellreceptors. Two distinct TNF receptors of approximately 55-kDa (TNFR1)and 75-kDa (TNFR2) have been identified [Hohman et al., J. Biol. Chem.,264:14927-14934 (1989); Brockhaus et al., Proc. Natl. Acad. Sci.,87:3127-3131 (1990); EP 417,563, published Mar. 20, 1991] and human andmouse cDNAs corresponding to both receptor types have been isolated andcharacterized [Loetscher et al., Cell, 61:351 (1990); Schall et al.,Cell, 61:361 (1990); Smith et al., Science, 248:1019-1023 (1990); Lewiset al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991); Goodwin et al.,Mol. Cell. Biol., 11:3020-3026 (1991)]. Extensive polymorphisms havebeen associated with both TNF receptor genes [see, e.g., Takao et al.,Immunogenetics, 37:199-203 (1993)]. Both TNFRs share the typicalstructure of cell surface receptors including extracellular,transmembrane and intracellular regions. The extracellular portions ofboth receptors are found naturally also as soluble TNF-binding proteins[Nophar, Y. et al., EMBO J., 9:3269 (1990); and Kohno, T. et al., Proc.Natl. Acad. Sci. U.S.A., 87:8331 (1990)]. The cloning of recombinantsoluble TNF receptors was reported by Hale et al. [J. Cell. Biochem.Supplement 15F, 1991, p. 113 (P424)].

The extracellular portion of type 1 and type 2 TNFRs (TNFR1 and TNFR2)contains a repetitive amino acid sequence pattern of four cysteine-richdomains (CRDs) designated 1 through 4, starting from the NH₂-terminus.Each CRD is about 40 amino acids long and contains 4 to 6 cysteineresidues at positions which are well conserved [Schall et al., supra;Loetscher et al., supra; Smith et al., supra; Nophar et al., supra;Kohno et al., supra]. In TNFR1, the approximate boundaries of the fourCRDs are as follows: CRD1—amino acids 14 to about 53; CRD2—amino acidsfrom about 54 to about 97; CRD3—amino acids from about 98 to about 138;CRD4—amino acids from about 139 to about 167. In TNFR2, CRD1 includesamino acids 17 to about 54; CRD2—amino acids from about 55 to about 97;CRD3—amino acids from about 98 to about 140; and CRD4—amino acids fromabout 141 to about 179 [Banner et al., Cell, 73:431-435 (1993)]. Thepotential role of the CRDs in ligand binding is also described by Banneret al., supra.

A similar repetitive pattern of CRDs exists in several othercell-surface proteins, including the p75 nerve growth factor receptor(NGFR) [Johnson et al., Cell, 47:545 (1986); Radeke et al., Nature,325:593 (1987)], the B cell antigen CD40 [Stamenkovic et al., EMBO J.,8:1403 (1989)], the T cell antigen OX40 [Mallet et al., EMBO J., 9:1063(1990)] and the Fas antigen [Yonehara et al., supra and Itoh et al.,supra]. CRDs are also found in the soluble TNFR (sTNFR)-like T2 proteinsof the Shope and myxoma poxviruses [Upton et al., Virology, 160:20-29(1987); Smith et al., Biochem. Biophys. Res. Commun., 176:335 (1991);Upton et al., Virology, 184:370 (1991)]. Optimal alignment of thesesequences indicates that the positions of the cysteine residues are wellconserved. These receptors are sometimes collectively referred to asmembers of the TNF/NGF receptor superfamily. Recent studies on p75NGFRshowed that the deletion of CRD1 [Welcher, A. A. et al., Proc. Natl.Acad. Sci. USA, 88:159-163 (1991)] or a 5-amino acid insertion in thisdomain [Yan, H. and Chao, M. V., J. Biol. Chem., 266:12099-12104 (1991)]had little or no effect on NGF binding [Yan, H. and Chao, M. V., supra].p75 NGFR contains a proline-rich stretch of about 60 amino acids,between its CRD4 and transmembrane region, which is not involved in NGFbinding [Peetre, C. et al., Eur. J. Hematol., 41:414-419 (1988);Seckinger, P. et al., J. Biol. Chem., 264:11966-11973 (1989); Yan, H.and Chao, M. V., supra]. A similar proline-rich region is found in TNFR2but not in TNFR1.

Itoh et al. disclose that the Apo-1 receptor can signal an apoptoticcell death similar to that signaled by the 55-kDa TNFR1 [Itoh et al.,supra]. Expression of the Apo-1 antigen has also been reported to bedown-regulated along with that of TNFR1 when cells are treated witheither TNF-α or anti-Apo-1 mouse monoclonal antibody [Krammer et al.,supra; Nagata et al., supra]. Accordingly, some investigators havehypothesized that cell lines that co-express both Apo-1 and TNFR1receptors may mediate cell killing through common signaling pathways[Id.].

The TNF family ligands identified to date, with the exception oflymphotoxin-α, are type II transmembrane proteins, whose C-terminus isextracellular. In contrast, the receptors in the TNF receptor (TNFR)family identified to date are type I transmembrane proteins. In both theTNF ligand and receptor families, however, homology identified betweenfamily members has been found mainly in the extracellular domain(“ECD”). Several of the TNF family cytokines, including TNF-α, Apo-1ligand and CD40 ligand, are cleaved proteolytically at the cell surface;the resulting protein in each case typically forms a homotrimericmolecule that functions as a soluble cytokine. TNF receptor familyproteins are also usually cleaved proteolytically to release solublereceptor ECDs that can function as inhibitors of the cognate cytokines.

Recently, other members of the mammalian TNFR family have beenidentified. In Marsters et al., Curr. Biol., 6:750 (1996), investigatorsdescribe a full length native sequence human polypeptide, called Apo-3,which exhibits similarity to the TNFR family in its extracellularcysteine-rich repeats and resembles TNFR1 and CD95 in that it contains acytoplasmic death domain sequence [see also Marsters et al., Curr.Biol., 6:1669 (1996)]. Apo-3 has also been referred to by otherinvestigators as DR3, wsl-1 and TRAMP [Chinnaiyan et al., Science,274:990 (1996); Kitson et al., Nature, 384:372 (1996); Bodmer et al.,Immunity, 6:79 (1997)].

Pan et al. have disclosed another TNF receptor family member referred toas “DR4” [Pan et al., Science, 276:111-113 (1997)]. The DR4 was reportedto contain a cytoplasmic death domain capable of engaging the cellsuicide apparatus. Pan et al. disclose that DR4 is believed to be areceptor for the ligand known as Apo-2 ligand or TRAIL.

The Apoptosis-Inducing Signaling Complex

As presently understood, the cell death program contains at least threeimportant elements—activators, inhibitors, and effectors; in C. elegans,these elements are encoded respectively by three genes, Ced-4, Ced-9 andCed-3 [Steller, Science, 267:1445 (1995); Chinnaiyan et al., Science,275:1122-1126 (1997)]. Two of the TNFR family members, TNFR1 andFas/Apo1 (CD95), can activate apoptotic cell death [Chinnaiyan andDixit, Current Biology, 6:555-562 (1996); Fraser and Evan, Cell;85:781-784 (1996)]. TNFR1 is also known to mediate activation of thetranscription factor, NF-κB [Tartaglia et al., Cell, 74:845-853 (1993);Hsu et al., Cell, 84:299-308 (1996)]. In addition to some ECD homology,these two receptors share homology in their intracellular domain (ICD)in an oligomerization interface known as the death domain [Tartaglia etal., supra; Nagata, Cell, 88:355 (1997)]. Death domains are also foundin several metazoan proteins that regulate apoptosis, namely, theDrosophila protein, Reaper, and the mammalian proteins referred to asFADD/MORT1, TRADD, and RIP [Cleaveland and Ihle, Cell, 81:479-482(1995)]. Using the yeast-two hybrid system, Raven et al. report theidentification of protein, wsl-1, which binds to the TNFR1 death domain[Raven et al., Programmed Cell Death Meeting, Sep. 20-24, 1995, Abstractat page 127; Raven et al., European Cytokine Network, 7:Abstr. 82 atpage 210 (April-June 1996)]. The wsl-1 protein is described as beinghomologous to TNFR1 (48% identity) and having a restricted tissuedistribution. According to Raven et al., the tissue distribution ofwsl-1 is significantly different from the TNFR1 binding protein, TRADD.

Upon ligand binding and receptor clustering, TNFR1 and CD95 are believedto recruit FADD into a death-inducing signalling complex. CD95purportedly binds FADD directly, while TNFR1 binds FADD indirectly viaTRADD [Chinnaiyan et al., Cell, 81:505-512 (1995); Boldin et al., J.Biol. Chem., 270:387-391 (1995); Hsu et al., supra; Chinnaiyan et al.,J. Biol. Chem., 271:4961-4965 (1996)]. It has been reported that FADDserves as an adaptor protein which recruits the Ced-3-related protease,MACHα/FLICE (caspase 8), into the death signalling complex [Boldin etal., Cell, 85:803-815 (1996); Muzio et al., Cell, 85:817-827 (1996)].MACHα/FLICE appears to be the trigger that sets off a cascade ofapoptotic proteases, including the interleukin-1β converting enzyme(ICE) and CPP32/Yama, which may execute some critical aspects of thecell death programme [Fraser and Evan, supra].

It was recently disclosed that programmed cell death involves theactivity of members of a family of cysteine proteases related to the C.elegans cell death gene, ced-3, and to the mammalian IL-1-convertingenzyme, ICE. The activity of the ICE and CPP32/Yama proteases can beinhibited by the product of the cowpox virus gene, crmA [Ray et al.,Cell, 69:597-604 (1992); Tewari et al., Cell, 81:801-809 (1995)]. Recentstudies show that CrmA can inhibit TNFR1- and CD95-induced cell death[Enari et al., Nature, 375:78-81 (1995); Tewari et al., J. Biol. Chem.,270:3255-3260 (1995)].

As reviewed recently by Tewari et al., TNFR1, TNFR2 and CD40 modulatethe expression of proinflammatory and costimulatory cytokines, cytokinereceptors, and cell adhesion molecules through activation of thetranscription factor, NF-κB [Tewari et al., Curr. Op. Genet. Develop.,6:39-44 (1996)]. NF-κB is the prototype of a family of dimerictranscription factors whose subunits contain conserved Rel regions[Verma et al., Genes Develop., 9:2723-2735 (1996); Baldwin, Ann. Rev.Immunol., 14:649-681 (1996)]. In its latent form, NF-κB is complexedwith members of the IκB inhibitor family; upon inactivation of the IκBin response to certain stimuli, released NF-κB translocates to thenucleus where it binds to specific DNA sequences and activates genetranscription.

For a review of the TNF family of cytokines and their receptors, seeGruss and Dower, supra.

SUMMARY OF THE INVENTION

Applicants have identified cDNA clones that encode novel polypeptides,designated in the present application as “Apo-2.” It is believed thatApo-2 is a member of the TNFR family; full-length native sequence humanApo-2 polypeptide exhibits some similarities to some known TNFRs,including a cytoplasmic death domain region. Full-length native sequencehuman Apo-2 also exhibits similarity to the TNFR family in itsextracellular cysteine-rich repeats. Apo-2 polypeptide has been found tobe capable of triggering caspase-dependent apoptosis and activatingNF-κB. Applicants surprisingly found that a soluble extracellular domainof Apo-2 binds Apo-2 ligand (“Apo-2L”) and can inhibit Apo-2 ligandfunction. It is presently believed that Apo-2 ligand can signal via atleast two different receptors, DR4 and the newly described Apo-2 herein.

In one embodiment, the invention provides isolated Apo-2 polypeptide. Inparticular, the invention provides isolated native sequence Apo-2polypeptide, which in one embodiment, includes an amino acid sequencecomprising residues 1 to 411 of FIG. 1 (SEQ ID NO:1). In otherembodiments, the isolated Apo-2 polypeptide comprises at least about 80%amino acid sequence identity with native sequence Apo-2 polypeptidecomprising residues 1 to 411 of FIG. 1 (SEQ ID NO:1). Optionally, theApo-2 polypeptide is obtained or obtainable by expressing thepolypeptide encoded by the cDNA insert of the vector deposited as ATCC209021.

In another embodiment, the invention provides an isolated extracellulardomain (ECD) sequence of Apo-2. Optionally, the isolated extracellulardomain sequence comprises amino acid residues 54 to 182 of FIG. 1 (SEQID NO:1).

In another embodiment, the invention provides an isolated death domainsequence of Apo-2. Optionally, the isolated death domain sequencecomprises amino acid residues 324 to 391 of FIG. 1 (SEQ ID NO:1).

In another embodiment, the invention provides chimeric moleculescomprising Apo-2 polypeptide fused to a heterologous polypeptide oramino acid sequence. An example of such a chimeric molecule comprises anApo-2 fused to an immunoglobulin sequence. Another example comprises anextracellular domain sequence of Apo-2 fused to a heterologouspolypeptide or amino acid sequence, such as an immunoglobulin sequence.

In another embodiment, the invention provides an isolated nucleic acidmolecule encoding Apo-2 polypeptide. In one aspect, the nucleic acidmolecule is RNA or DNA that encodes an Apo-2 polypeptide or a particulardomain of Apo-2, or is complementary to such encoding nucleic acidsequence, and remains stably bound to it under at least moderate, andoptionally, under high stringency conditions. Such complementary nucleicacid may be fully complementary to the entire length of the RNA or DNA.It is contemplated that the complementary nucleic acid may also becomplementary to only a fragment of the RNA or DNA nucleotide sequence.In one embodiment, the nucleic acid sequence is selected from:

-   -   (a) the coding region of the nucleic acid sequence of FIG. 1        (SEQ ID NO:2) that codes for residue 1 to residue 411 (i.e.,        nucleotides 140-142 through 1370-1372), inclusive;    -   (b) the coding region of the nucleic acid sequence of FIG. 1        (SEQ ID NO:2) that codes for residue 1 to residue 182 (i.e.,        nucleotides 140-142 through 683-685), inclusive;    -   (c) the coding region of the nucleic acid sequence of FIG. 1        (SEQ ID NO:2) that codes for residue 54 to residue 182 (i.e.,        nucleotides 299-301 through 683-685), inclusive;    -   (d) the coding region of the nucleic acid sequence of FIG. 1        (SEQ ID NO:2) that codes for residue 324 to residue 391 (i.e.,        nucleotides 1109-1111 through 1310-1312), inclusive; or    -   (e) a sequence corresponding to the sequence of (a), (b), (c)        or (d) within the scope of degeneracy of the genetic code. The        isolated nucleic acid may comprise the Apo-2 polypeptide cDNA        insert of the vector deposited as ATCC 209021 which includes the        nucleotide sequence encoding Apo-2 polypeptide.

In a further embodiment, the invention provides a vector comprising thenucleic acid molecule encoding the Apo-2 polypeptide or particulardomain of Apo-2. A host cell comprising the vector or the nucleic acidmolecule is also provided. A method of producing Apo-2 is furtherprovided.

In another embodiment, the invention provides an antibody whichspecifically binds to Apo-2. The antibody may be an agonistic,antagonistic or neutralizing antibody. Single-chain antibodies anddimeric molecules, in particular homodimeric molecules, comprising Apo-2antibody are also provided.

In another embodiment, the invention provides non-human, transgenic orknock-out animals.

A further embodiment of the invention provides articles of manufactureand kits that include Apo-2 or Apo-2 antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of a native sequence human Apo-2cDNA (SEQ ID NO:2) and its derived amino acid sequence (SEQ ID NO:1).

FIG. 2A shows the derived amino acid sequence of a native sequence humanApo-2—the putative signal sequence is underlined, the putativetransmembrane domain is boxed, and the putative death domain sequence isdash underlined. The cysteines of the two cysteine-rich domains areindividually underlined.

FIG. 2B shows an alignment and comparison of the death domain sequencesof native sequence human Apo-2, DR4, Apo-3/DR3, TNFR1, and Fas/Apo-1(CD95). Asterisks indicate residues that are essential for deathsignaling by TNFR1 [Tartaglia et al., supra].

FIG. 3 shows the interaction of the Apo-2 ECD with Apo-2L. Supernatantsfrom mock-transfected 293 cells or from 293 cells transfected with Flagepitope-tagged Apo-2 ECD were incubated with poly-His-tagged Apo-2L andsubjected to immunoprecipitation with anti-Flag conjugated or Nickelconjugated agarose beads. The precipitated proteins were resolved byelectrophoresis on polyacrylamide gels, and detected by immunoblot withanti-Apo-2L or anti-Flag antibody.

FIG. 4 shows the induction of apoptosis by Apo-2 and inhibition ofApo-2L activity by soluble Apo-2 ECD. Human 293 cells (A, B) or HeLacells (C) were transfected by pRK5 vector or by pRK5-based plasmidsencoding Apo-2 and/or CrmA. Apoptosis was assessed by morphology (A),DNA fragmentation (B), or by FACS (C-E). Soluble Apo-2L waspre-incubated with buffer or affinity-purified Apo-2 ECD together withanti-Flag antibody or Apo-2 ECD immunoadhesin or DR4 or TNFR1immunoadhesins and added to HeLa cells. The cells were later analyzedfor apoptosis (D). Dose-response analysis using Apo-2L with Apo-2 ECDimmunoadhesin was also determined (E).

FIG. 5 shows activation of NF-κB by Apo-2, DR4, and Apo-2L. (A) HeLacells were transfected with expression plasmids encoding the indicatedproteins. Nuclear extracts were prepared and analyzed by anelectrophoretic mobility shift assay. (B) HeLa cells or MCF7 cells weretreated with buffer, Apo-2L or TNF-alpha and assayed for NF-κB activity.(C) HeLa cells were preincubated with buffer, ALLN or cyclohexamidebefore addition of Apo-2L. Apoptosis was later analyzed by FACS.

FIG. 6A shows expression of Apo-2 mRNA in human tissues as analyzed byNorthern hybridization of human tissue poly A RNA blots.

FIG. 6B shows expression of Apo-2 mRNA in human cancer cell lines asanalyzed by Northern hybridization of human cancer cell line poly A RNAblots.

FIG. 7 shows the FACS analysis of an Apo-2 antibody, 3F11.39.7(illustrated by the bold lines) as compared to IgG controls (dottedlines). The 3F11.39.7 antibody recognized the Apo-2 receptor expressedin human 9D cells.

FIG. 8 is a graph showing percent (%) apoptosis induced in 9D cells byApo-2 antibody 3F11.39.7, in the absence of goat anti-mouse IgG Fc.

FIG. 9 is a bar diagram showing percent (%) apoptosis, as compared toApo-2L, in 9D cells by Apo-2 antibody 3F11.39.7 in the presence orabsence of goat anti-mouse IgG Fc.

FIG. 10 is a bar diagram illustrating the ability of Apo-2 antibody3F11.39.7 to block the apoptosis induced by Apo-2L in 9D cells.

FIG. 11 is a graph showing results of an ELISA testing binding of Apo-2antibody 3F11.39.7 to Apo-2 and to other known Apo-2L receptors referredto as DR4, DcR1, and DcR2.

FIG. 12A is a graph showing the results of an ELISA assay evaluatingbinding of the 16E2 antibody to Apo-2, DR4, DcR1, DcR2 and CD4-Ig.

FIG. 12B is a graph showing the results of an ELISA assay evaluatingbinding of the 20E6 antibody to Apo-2, DR4, DcR1, DcR2 and CD4-Ig.

FIG. 12C is a graph showing the results of an ELISA assay evaluatingbinding of the 24C4 antibody to Apo-2, DR4, DcR1, DcR2 and CD4-Ig.

FIG. 13A is a graph showing agonistic activity of the 16E2 antibody, ascompared to Apo-2L, in an apoptosis assay (crystal violet stain) usingSK-MES-1 cells.

FIG. 13B is a bar diagram showing agonistic activity of the 16E2antibody, as compared to 7D5 scFv antibody (an anti-tissue factorantibody), in an apoptosis assay (crystal violet stain) using SK-MES-1cells.

FIG. 13C is a bar diagram showing agonistic activity of the 16E2antibody, as compared to 7D5 scFv antibody, in an apoptosis assay(annexin V-biotin/streptavidin-[S³⁵]) using SK-MES-1 cells.

FIG. 14A is a graph showing agonistic activity of the 20E6 antibody, ascompared to Apo-2L, in an apoptosis assay (crystal violet stain) usingSK-MES-1 cells.

FIG. 14B is a graph showing agonistic activity of the 20E6 antibody by acomparison between results obtained in the crystal violet and annexinV-biotin/streptavidin-[S³⁵] apoptosis assays.

FIG. 14C is a graph showing agonistic activity of gD-tagged 16E2antibody, as compared to Apo-2L, in an apoptosis assay (crystal violetstain) using SK-MES-1 cells

FIG. 15A shows the nucleotide sequence of the single chain antibody(scFv) fragment referred to as 16E2 (SEQ ID NO:6).

FIG. 15B shows the nucleotide sequence of the single chain antibody(scFv) fragment referred to as 20E6 (SEQ ID NO:7).

FIG. 15C shows the nucleotide sequence of the single chain antibody(scFv) fragment referred to as 24C4 (SEQ ID NO:8).

FIG. 16 shows the single chain antibody (scFv) fragments referred to as16E2, 20E6 and 24C4, with the respective amino acid sequences for thesignal sequence and the heavy and light chain CDR regions identified(CDR1, CDR2, and CDR3 regions are underlined).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The terms “Apo-2 polypeptide” and “Apo-2” when used herein encompassnative sequence Apo-2 and Apo-2 variants (which are further definedherein). These terms encompass Apo-2 from a variety of mammals,including humans. The Apo-2 may be isolated from a variety of sources,such as from human tissue types or from another source, or prepared byrecombinant or synthetic methods.

A “native sequence Apo-2” comprises a polypeptide having the same aminoacid sequence as an Apo-2 derived from nature. Thus, a native sequenceApo-2 can have the amino acid sequence of naturally-occurring Apo-2 fromany mammal. Such native sequence Apo-2 can be isolated from nature orcan be produced by recombinant or synthetic means. The term “nativesequence Apo-2” specifically encompasses naturally-occurring truncatedor secreted forms of the Apo-2 (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the Apo-2. Anaturally-occurring variant form of the Apo-2 includes an Apo-2 havingan amino acid substitution at residue 410 in the amino acid sequenceshown in FIG. 1 (SEQ ID NO:1). In one embodiment of suchnaturally-occurring variant form, the leucine residue at position 410 issubstituted by a methionine residue. In FIG. 1 (SEQ ID NO:1), the aminoacid residue at position 410 is identified as “Xaa” to indicate that theamino acid may, optionally, be either leucine or methionine. In FIG. 1(SEQ ID NO:2), the nucleotide at position 1367 is identified as “W” toindicate that the nucleotide may be either adenine (A) or thymine (T) oruracil (U). In one embodiment of the invention, the native sequenceApo-2 is a mature or full-length native sequence Apo-2 comprising aminoacids 1 to 411 of FIG. 1 (SEQ ID NO:1). Optionally, the Apo-2 isobtained or obtainable by expressing the polypeptide encoded by the cDNAinsert of the vector deposited as ATCC 209021.

The “Apo-2 extracellular domain” or “Apo-2 ECD” refers to a form ofApo-2 which is essentially free of the transmembrane and cytoplasmicdomains of Apo-2. Ordinarily, Apo-2 ECD will have less than 1% of suchtransmembrane and/or cytoplasmic domains and preferably, will have lessthan 0.5% of such domains. Optionally, Apo-2 ECD will comprise aminoacid residues 54 to 182 of FIG. 1 (SEQ ID NO:1) or amino acid residues 1to 182 of FIG. 1 (SEQ ID NO:1). Optionally, Apo-2 ECD will comprise oneor more cysteine-rich domains, and preferably, one or both of thecysteine-rich domains identified herein (see FIG. 2A). It will beunderstood by the skilled artisan that the transmembrane domainidentified for the Apo-2 polypeptide herein is identified pursuant tocriteria routinely employed in the art for identifying that type ofhydrophobic domain. The exact boundaries of a transmembrane domain mayvary but most likely by no more than about 5 amino acids at either endof the domain specifically mentioned herein.

“Apo-2 variant” means a biologically active Apo-2 as defined belowhaving at least about 80% amino acid sequence identity with the Apo-2having the deduced amino acid sequence shown in FIG. 1 (SEQ ID NO:1) fora full-length native sequence human Apo-2 or the sequences identifiedherein for Apo-2 ECD or death domain. Such Apo-2 variants include, forinstance, Apo-2 polypeptides wherein one or more amino acid residues areadded, or deleted, at the N- or C-terminus of the sequence of FIG. 1(SEQ ID NO:1) or the sequences identified herein for Apo-2 ECD or deathdomain. Ordinarily, an Apo-2 variant will have at least about 80% aminoacid sequence identity, more preferably at least about 90% amino acidsequence identity, and even more preferably at least about 95% aminoacid sequence identity with the amino acid sequence of FIG. 1 (SEQ IDNO:1) or the sequences identified herein for Apo-2 ECD or death domain.

“Percent (%) amino acid sequence identity” with respect to the Apo-2sequences identified herein is defined as the percentage of amino acidresidues in a candidate sequence that are identical with the amino acidresidues in the Apo-2 sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as ALIGN™ or Megalign (DNASTAR) software. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising Apo-2 or Apo-2 antibody, or a domain sequencethereof, fused to a “tag polypeptide”. The tag polypeptide has enoughresidues to provide an epitope against which an antibody can be made,yet is short enough such that it does not interfere with activity of theApo-2 or Apo-2 antibody. The tag polypeptide preferably also is fairlyunique so that the antibody does not substantially cross-react withother epitopes. Suitable tag polypeptides generally have at least sixamino acid residues and usually between about 8 to about 50 amino acidresidues (preferably, between about 10 to about 20 residues).

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the Apo-2 naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” Apo-2 nucleic acid molecule is a nucleic acid moleculethat is identified and separated from at least one contaminant nucleicacid molecule with which it is ordinarily associated in the naturalsource of the Apo-2 nucleic acid. An isolated Apo-2 nucleic acidmolecule is other than in the form or setting in which it is found innature. Isolated Apo-2 nucleic acid molecules therefore aredistinguished from the Apo-2 nucleic acid molecule as it exists innatural cells. However, an isolated Apo-2 nucleic acid molecule includesApo-2 nucleic acid molecules contained in cells that ordinarily expressApo-2 where, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers anti-Apo-2 monoclonal antibodies (including agonist, antagonist,and blocking or neutralizing antibodies) and anti-Apo-2 antibodycompositions with polyepitopic specificity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-Apo-2 antibody with a constant domain, or a lightchain with a heavy chain, or a chain from one species with a chain fromanother species, or fusions with heterologous proteins, regardless ofspecies of origin or immunoglobulin class or subclass designation, aswell as antibody fragments (e.g., Fab, F(ab′)₂, and Fv), so long as theyexhibit the desired biological activity. See, e.g. U.S. Pat. No.4,816,567 and Mage et al., in Monoclonal Antibody Production Techniquesand Applications, pp. 79-97 (Marcel Dekker, Inc.: New York, 1987).

Thus, the modifier “monoclonal” indicates the character of the antibodyas being obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,Nature, 256:495 (1975), or may be made by recombinant DNA methods suchas described in U.S. Pat. No. 4,816,567. The “monoclonal antibodies” mayalso be isolated from phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990), for example.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies,vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.269-315 (1994). The scFv antibody fragments of the present inventioninclude but are not limited to the 16E2, 20E6 and 24C4 antibodiesdescribed in detail below. Within the scope of the scFv antibodies ofthe invention are scFv antibodies comprising VH and VL domains thatinclude one or more of the CDR regions identified for the 16E2, 20E6 and24C4 antibodies.

“Humanized” forms of non-human (e.g. murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains, or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, the humanized antibody may comprise residues which arefound neither in the recipient antibody nor in the imported CDR orframework sequences. These modifications are made to further refine andoptimize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region or domain (Fc), typically that of ahuman immunoglobulin.

“Biologically active” and “desired biological activity” for the purposesherein means (1) having the ability to modulate apoptosis (either in anagonistic or stimulating manner or in an antagonistic or blockingmanner) in at least one type of mammalian cell in vivo or ex vivo; (2)having the ability to bind Apo-2 ligand; or (3) having the ability tomodulate Apo-2 ligand signaling and Apo-2 ligand activity.

The terms “apoptosis” and “apoptotic activity” are used in a broad senseand refer to the orderly or controlled form of cell death in mammalsthat is typically accompanied by one or more characteristic cellchanges, including condensation of cytoplasm, loss of plasma membranemicrovilli, segmentation of the nucleus, degradation of chromosomal DNAor loss of mitochondrial function. This activity can be determined andmeasured, for instance, by cell viability assays, FACS analysis or DNAelectrophoresis, all of which are known in the art.

The terms “treating,” “treatment,” and “therapy” as used herein refer tocurative therapy, prophylactic therapy, and preventative therapy.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, blastoma,gastrointestinal cancer, renal cancer, pancreatic cancer, glioblastoma,neuroblastoma, cervical cancer, ovarian cancer, liver cancer, stomachcancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial carcinoma, salivary gland carcinoma,kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer.

The term “mammal” as used herein refers to any mammal classified as amammal, including humans, cows, horses, dogs and cats. In a preferredembodiment of the invention, the mammal is a human.

II. Compositions and Methods of the Invention

The present invention provides newly identified and isolated Apo-2polypeptides and Apo-2 antibodies. In particular, Applicants haveidentified and isolated various human Apo-2 polypeptides. The propertiesand characteristics of some of these Apo-2 polypeptides and anti-Apo-2antibodies are described in further detail in the Examples below. Basedupon the properties and characteristics of the Apo-2 polypeptidesdisclosed herein, it is Applicants' present belief that Apo-2 is amember of the TNFR family.

A description follows as to how Apo-2, as well as Apo-2 chimericmolecules and anti-Apo-2 antibodies, may be prepared.

A. Preparation of Apo-2

The description below relates primarily to production of Apo-2 byculturing cells transformed or transfected with a vector containingApo-2 nucleic acid. It is of course, contemplated that alternativemethods, which are well known in the art, may be employed to prepareApo-2.

1. Isolation of DNA Encoding Apo-2

The DNA encoding Apo-2 may be obtained from any cDNA library preparedfrom tissue believed to possess the Apo-2 mRNA and to express it at adetectable level. Accordingly, human Apo-2 DNA can be convenientlyobtained from a cDNA library prepared from human tissues, such as thebacteriophage libraries of human pancreas and kidney cDNA described inExample 1. The Apo-2-encoding gene may also be obtained from a genomiclibrary or by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to the Apo-2or oligonucleotides of at least about 20-80 bases) designed to identifythe gene of interest or the protein encoded by it. Screening the cDNA orgenomic library with the selected probe may be conducted using standardprocedures, such as described in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).An alternative means to isolate the gene encoding Apo-2 is to use PCRmethodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

A preferred method of screening employs selected oligonucleotidesequences to screen cDNA libraries from various human tissues. Example 1below describes techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Nucleic acid having all the protein coding sequence may be obtained byscreening selected cDNA or genomic libraries using the deduced aminoacid sequence disclosed herein for the first time, and, if necessary,using conventional primer extension procedures as described in Sambrooket al., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

Apo-2 variants can be prepared by introducing appropriate nucleotidechanges into the Apo-2 DNA, or by synthesis of the desired Apo-2polypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the Apo-2, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the native full-length sequence Apo-2 or in variousdomains of the Apo-2 described herein, can be made, for example, usingany of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the Apo-2 that results in a change in theamino acid sequence of the Apo-2 as compared with the native sequenceApo-2. Optionally the variation is by substitution of at least one aminoacid with any other amino acid in one or more of the domains of theApo-2 molecule. The variations can be made using methods known in theart such as oligonucleotide-mediated (site-directed) mutagenesis,alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carteret al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. AcidsRes., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315(1985)], restriction selection mutagenesis [Wells et al., Philos. Trans.R. Soc. London SerA, 317:415 (1986)] or other known techniques can beperformed on the cloned DNA to produce the Apo-2 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence which are involved in theinteraction with a particular ligand or receptor. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine isthe preferred scanning amino acid among this group because it eliminatesthe side-chain beyond the beta-carbon and is less likely to alter themain-chain conformation of the variant. Alanine is also preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

Once selected Apo-2 variants are produced, they can be contacted with,for instance, Apo-2L, and the interaction, if any, can be determined.The interaction between the Apo-2 variant and Apo-2L can be measured byan in vitro assay, such as described in the Examples below. While anynumber of analytical measurements can be used to compare activities andproperties between a native sequence Apo-2 and an Apo-2 variant, aconvenient one for binding is the dissociation constant K_(d) of thecomplex formed between the Apo-2 variant and Apo-2L as compared to theK_(d) for the native sequence Apo-2. Generally, a ≧3-fold increase ordecrease in K_(d) per substituted residue indicates that the substitutedresidue(s) is active in the interaction of the native sequence Apo-2with the Apo-2L.

Optionally, representative sites in the Apo-2 sequence suitable formutagenesis would include sites within the extracellular domain, andparticularly, within one or both of the cysteine-rich domains. Suchvariations can be accomplished using the methods described above.

2. Insertion of Nucleic Acid into a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding Apo-2 may beinserted into a replicable vector for further cloning (amplification ofthe DNA) or for expression. Various vectors are publicly available. Thevector components generally include, but are not limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, an enhancer element, a promoter, and a transcriptiontermination sequence, each of which is described below.

(i) Signal Sequence Component

The Apo-2 may be produced recombinantly not only directly, but also as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe Apo-2 DNA that is inserted into the vector. The heterologous signalsequence selected preferably is one that is recognized and processed(i.e., cleaved by a signal peptidase) by the host cell. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin IT leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), orthe signal described in WO 90/13646 published Nov. 15, 1990. Inmammalian cell expression the native Apo-2 presequence that normallydirects insertion of Apo-2 in the cell membrane of human cells in vivois satisfactory, although other mammalian signal sequences may be usedto direct secretion of the protein, such as signal sequences fromsecreted polypeptides of the same or related species, as well as viralsecretory leaders, for example, the herpes simplex glycoprotein Dsignal.

The DNA for such precursor region is preferably ligated in reading frameto DNA encoding Apo-2.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used because it contains the earlypromoter).

Most expression vectors are “shuttle” vectors, i.e., they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of Apo-2 DNA. However, the recovery of genomic DNA encodingApo-2 is more complex than that of an exogenously replicated vectorbecause restriction enzyme digestion is required to excise the Apo-2DNA.

(iii) Selection Gene Component

Expression and cloning vectors typically contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin [Southern et al., J. Molec. Appl. Genet., 1:327(1982)], mycophenolic acid (Mulligan et al., Science, 209:1422 (1980)]or hygromycin [Sugden et al., Mol. Cell. Biol., 5:410-413 (1985)]. Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theApo-2 nucleic acid, such as DHFR or thymidine kinase. The mammalian celltransformants are placed under selection pressure that only thetransformants are uniquely adapted to survive by virtue of having takenup the marker. Selection pressure is imposed by culturing thetransformants under conditions in which the concentration of selectionagent in the medium is successively changed, thereby leading toamplification of both the selection gene and the DNA that encodes Apo-2.Amplification is the process by which genes in greater demand for theproduction of a protein critical for growth are reiterated in tandemwithin the chromosomes of successive generations of recombinant cells.Increased quantities of Apo-2 are synthesized from the amplified DNA.Other examples of amplifiable genes include metallothionein-I and -II,adenosine deaminase, and ornithine decarboxylase.

Cells transformed with the DHFR selection gene may first be identifiedby culturing all of the transformants in a culture medium that containsmethotrexate (Mtx), a competitive antagonist of DHFR. An appropriatehost cell when wild-type DHFR is employed is the Chinese hamster ovary(CHO) cell line deficient in DHFR activity, prepared and propagated asdescribed by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980).The transformed cells are then exposed to increased levels ofmethotrexate. This leads to the synthesis of multiple copies of the DHFRgene, and, concomitantly, multiple copies of other DNA comprising theexpression vectors, such as the DNA encoding Apo-2. This amplificationtechnique can be used with any otherwise suitable host, e.g., ATCC No.CCL61 CHO-K1, notwithstanding the presence of endogenous DHFR if, forexample, a mutant DHFR gene that is highly resistant to Mtx is employed(EP 117,060).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding Apo-2, wild-type DHFR protein, and another selectable markersuch as aminoglycoside 3′-phosphotransferase (APH) can be selected bycell growth in medium containing a selection agent for the selectablemarker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin,or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979);Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157(1980)]. The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)]. The presence of thetrp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts [Bianchi et al.,Curr. Genet., 12:185 (1987)]. More recently, an expression system forlarge-scale production of recombinant calf chymosin was reported for K.lactis [Van den Berg, Bio/Technology, 8:135 (1990)]. Stable multi-copyexpression vectors for secretion of mature recombinant human serumalbumin by industrial strains of Kluyveromyces have also been disclosed[Fleer et al., Bio/Technology, 9:968-975 (1991)].

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the Apo-2nucleic acid sequence. Promoters are untranslated sequences locatedupstream (5′) to the start codon of a structural gene (generally withinabout 100 to 1000 bp) that control the transcription and translation ofparticular nucleic acid sequence, such as the Apo-2 nucleic acidsequence, to which they are operably linked. Such promoters typicallyfall into two classes, inducible and constitutive. Inducible promotersare promoters that initiate increased levels of transcription from DNAunder their control in response to some change in culture conditions,e.g., the presence or absence of a nutrient or a change in temperature.At this time a large number of promoters recognized by a variety ofpotential host cells are well known. These promoters are operably linkedto Apo-2 encoding DNA by removing the promoter from the source DNA byrestriction enzyme digestion and inserting the isolated promotersequence into the vector. Both the native Apo-2 promoter sequence andmany heterologous promoters may be used to direct amplification and/orexpression of the Apo-2 DNA.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems [Chang et al., Nature, 275:615(1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, atryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057(1980); EP 36,776], and hybrid promoters such as the tac promoter[deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. However,other known bacterial promoters are suitable. Their nucleotide sequenceshave been published, thereby enabling a skilled worker operably toligate them to DNA encoding Apo-2 [Siebenlist et al., Cell, 20:269(1980)] using linkers or adaptors to supply any required restrictionsites. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingApo-2.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Apo-2 transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter, fromheat-shock promoters, and from the promoter normally associated with theApo-2 sequence, provided such promoters are compatible with the hostcell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication [Fiers et al., Nature, 273:113 (1978); Mulligan and Berg,Science, 209:1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci.USA, 78:7398-7402 (1981)]. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment [Greenaway et al., Gene, 18:355-360 (1982)]. A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978 [See also Gray et al.,Nature, 295:503-508 (1982) on expressing cDNA encoding immune interferonin monkey cells; Reyes et al., Nature, 297:598-601 (1982) on expressionof human β-interferon cDNA in mouse cells under the control of athymidine kinase promoter from herpes simplex virus; Canaani and Berg,Proc. Natl. Acad. Sci. USA 79:5166-5170 (1982) on expression of thehuman interferon gene in cultured mouse and rabbit cells; and Gorman etal., Proc. Natl. Acad. Sci. USA, 79:6777-6781 (1982) on expression ofbacterial CAT sequences in CV-1 monkey kidney cells, chicken embryofibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3cells using the Rous sarcoma virus long terminal repeat as a promoter].

(v) Enhancer Element Component

Transcription of a DNA encoding the Apo-2 of this invention by highereukaryotes may be increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription.Enhancers are relatively orientation and position independent, havingbeen found 5′ [Laimins et al., Proc. Natl. Acad. Sci. USA, 78:993(1981]) and 3′ [Lusky et al., Mol. Cell Bio., 3:1108 (1983]) to thetranscription unit, within an intron [Banerji et al., Cell, 33:729(1983)], as well as within the coding sequence itself [Osborne et al.,Mol. Cell Bio., 4:1293 (1984)]. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature, 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theApo-2 coding sequence, but is preferably located at a site 5′ from thepromoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding Apo-2.

(vii) Construction and Analysis of Vectors

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures can be used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9:309 (1981) or by the method of Maxim et al., Methods inEnzymology, 65:499 (1980).

(viii) Transient Expression Vectors

Expression vectors that provide for the transient expression inmammalian cells of DNA encoding Apo-2 may be employed. In general,transient expression involves the use of an expression vector that isable to replicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector [Sambrook et al., supra]. Transient expressionsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded bycloned DNAs, as well as for the rapid screening of such polypeptides fordesired biological or physiological properties. Thus, transientexpression systems are particularly useful in the invention for purposesof identifying Apo-2 variants.

(ix) Suitable Exemplary Vertebrate Cell Vectors

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of Apo-2 in recombinant vertebrate cell culture are describedin Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature,281:40-46 (1979); EP 117,060; and EP 117,058.

3. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include but are not limitedto eubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis (e.g., B.licheniformis 41P disclosed in DD 266,710 published Apr. 12, 1989),Pseudomonas such as P. aeruginosa, and Streptomyces. Preferably, thehost cell should secrete minimal amounts of proteolytic enzymes.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forApo-2-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein.

Suitable host cells for the expression of glycosylated Apo-2 are derivedfrom multicellular organisms. Such host cells are capable of complexprocessing and glycosylation activities. In principle, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified [See, e.g., Luckow et al., Bio/Technology, 6:47-55(1988); Miller et al., in Genetic Engineering, Setlow et al., eds., Vol.8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature,315:592-594 (1985)]. A variety of viral strains for transfection arepublicly available, e.g., the L-1 variant of Autographa californica NPVand the Bm-5 strain of Bombyx mori NPV.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens. During incubation of the plant cell culturewith A. tumefaciens, the DNA encoding the Apo-2 can be transferred tothe plant cell host such that it is transfected, and will, underappropriate conditions, express the Apo-2-encoding DNA. In addition,regulatory and signal sequences compatible with plant cells areavailable, such as the nopaline synthase promoter and polyadenylationsignal sequences [Depicker et al., J. Mol. Appl. Gen., 1:561 (1982)]. Inaddition, DNA segments isolated from the upstream region of the T-DNA780 gene are capable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue [EP321,196 published Jun. 21, 1989].

Propagation of vertebrate cells in culture (tissue culture) is also wellknown in the art [See, e.g., Tissue Culture, Academic Press, Kruse andPatterson, editors (1973)]. Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.,383:44-68 (1982)); MRC 5 cells; and FS4 cells.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors for Apo-2 production andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in Sambrook et al., supra, orelectroporation is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 publishedJun. 29, 1989. In addition, plants may be transfected using ultrasoundtreatment as described in WO 91/00358 published Jan. 10, 1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) is preferred. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

4. Culturing the Host Cells

Prokaryotic cells used to produce Apo-2 may be cultured in suitablemedia as described generally in Sambrook et al., supra.

The mammalian host cells used to produce Apo-2 may be cultured in avariety of media. Examples of commercially available media include Ham'sF10 (Sigma), Minimal Essential Medium (“MEM”, Sigma), RPMI-1640 (Sigma),and Dulbecco's Modified Eagle's Medium (“DMEM”, Sigma). Any such mediamay be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleosides (such as adenosine and thymidine),antibiotics (such as Gentamycin™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRLPress, 1991).

The host cells referred to in this disclosure encompass cells in cultureas well as cells that are within a host animal.

5. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, and particularly ³²P. However, other techniques may alsobe employed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionucleotides, fluorescers or enzymes. Alternatively,antibodies may be employed that can recognize specific duplexes,including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes orDNA-protein duplexes. The antibodies in turn may be labeled and theassay may be carried out where the duplex is bound to a surface, so thatupon the formation of duplex on the surface, the presence of antibodybound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. With immunohistochemicalstaining techniques, a cell sample is prepared, typically by dehydrationand fixation, followed by reaction with labeled antibodies specific forthe gene product coupled, where the labels are usually visuallydetectable, such as enzymatic labels, fluorescent labels, or luminescentlabels.

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a native sequence Apo-2 polypeptide or against a syntheticpeptide based on the DNA sequences provided herein or against exogenoussequence fused to Apo-2 DNA and encoding a specific antibody epitope.

6. Purification of Apo-2 Polypeptide

Forms of Apo-2 may be recovered from culture medium or from host celllysates. If the Apo-2 is membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g. Triton-X 100) or itsextracellular domain may be released by enzymatic cleavage.

When Apo-2 is produced in a recombinant cell other than one of humanorigin, the Apo-2 is free of proteins or polypeptides of human origin.However, it may be desired to purify Apo-2 from recombinant cellproteins or polypeptides to obtain preparations that are substantiallyhomogeneous as to Apo-2. As a first step, the culture medium or lysatemay be centrifuged to remove particulate cell debris. Apo-2 thereafteris purified from contaminant soluble proteins and polypeptides, with thefollowing procedures being exemplary of suitable purificationprocedures: by fractionation on an ion-exchange column; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on acation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel filtration using, for example, Sephadex G-75;and protein A Sepharose columns to remove contaminants such as IgG.

Apo-2 variants in which residues have been deleted, inserted, orsubstituted can be recovered in the same fashion as native sequenceApo-2, taking account of changes in properties occasioned by thevariation. For example, preparation of an Apo-2 fusion with anotherprotein or polypeptide, e.g., a bacterial or viral antigen,immunoglobulin sequence, or receptor sequence, may facilitatepurification; an immunoaffinity column containing antibody to thesequence can be used to adsorb the fusion polypeptide. Other types ofaffinity matrices also can be used.

A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) alsomay be useful to inhibit proteolytic degradation during purification,and antibiotics may be included to prevent the growth of adventitiouscontaminants. One skilled in the art will appreciate that purificationmethods suitable for native sequence Apo-2 may require modification toaccount for changes in the character of Apo-2 or its variants uponexpression in recombinant cell culture.

7. Covalent Modifications of Apo-2 Polypeptides

Covalent modifications of Apo-2 are included within the scope of thisinvention. One type of covalent modification of the Apo-2 is introducedinto the molecule by reacting targeted amino acid residues of the Apo-2with an organic derivatizing agent that is capable of reacting withselected side chains or the N- or C-terminal residues of the Apo-2.

Derivatization with bifunctional agents is useful for crosslinking Apo-2to a water-insoluble support matrix or surface for use in the method forpurifying anti-Apo-2 antibodies, and vice-versa. Derivatization with oneor more bifunctional agents will also be useful for crosslinking Apo-2molecules to generate Apo-2 dimers. Such dimers may increase bindingavidity and extend half-life of the molecule in vivo. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidyl-propionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)-dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group. The modifiedforms of the residues fall within the scope of the present invention.

Another type of covalent modification of the Apo-2 polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence Apo-2, and/oradding one or more glycosylation sites that are not present in thenative sequence Apo-2.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxylamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the Apo-2 polypeptide may beaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thenative sequence Apo-2 (for O-linked glycosylation sites). The Apo-2amino acid sequence may optionally be altered through changes at the DNAlevel, particularly by mutating the DNA encoding the Apo-2 polypeptideat preselected bases such that codons are generated that will translateinto the desired amino acids. The DNA mutation(s) may be made usingmethods described above and in U.S. Pat. No. 5,364,934, supra.

Another means of increasing the number of carbohydrate moieties on theApo-2 polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Depending on the coupling mode used, the sugar(s) maybe attached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the Apo-2 polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. For instance, chemical deglycosylation by exposing thepolypeptide to the compound trifluoromethanesulfonic acid, or anequivalent compound can result in the cleavage of most or all sugarsexcept the linking sugar (N-acetylglucosamine or N-acetylgalactosamine),while leaving the polypeptide intact. Chemical deglycosylation isdescribed by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987)and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavageof carbohydrate moieties on polypeptides can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.,Meth. Enzymol., 138:350 (1987).

Glycosylation at potential glycosylation sites may be prevented by theuse of the compound tunicamycin as described by Duksin et al., J. Biol.Chem., 257:3105 (1982). Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of Apo-2 comprises linking theApo-2 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

8. Apo-2 Chimeras

The present invention also provides chimeric molecules comprising Apo-2fused to another, heterologous polypeptide or amino acid sequence.

In one embodiment, the chimeric molecule comprises a fusion of the Apo-2with a tag polypeptide which provides an epitope to which an anti-tagantibody can selectively bind. The epitope tag is generally placed atthe amino- or carboxyl-terminus of the Apo-2. The presence of suchepitope-tagged forms of the Apo-2 can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe Apo-2 to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include the flu HA tag polypeptide and its antibody12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myctag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan etal., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al.,Protein Engineering, 3 (6):547-553 (1990)1. Other tag polypeptidesinclude the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210(1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194(1992)]; an α-tubulin epitope peptide [Skinner et al., J. Biol. Chem.,266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag[Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397(1990)]. Once the tag polypeptide has been selected, an antibody theretocan be generated using the techniques disclosed herein.

Generally, epitope-tagged Apo-2 may be constructed and producedaccording to the methods described above. Epitope-tagged Apo-2 is alsodescribed in the Examples below. Apo-2-tag polypeptide fusions arepreferably constructed by fusing the cDNA sequence encoding the Apo-2portion in-frame to the tag polypeptide DNA sequence and expressing theresultant DNA fusion construct in appropriate host cells. Ordinarily,when preparing the Apo-2-tag polypeptide chimeras of the presentinvention, nucleic acid encoding the Apo-2 will be fused at its 3′ endto nucleic acid encoding the N-terminus of the tag polypeptide, however5′ fusions are also possible. For example, a polyhistidine sequence ofabout 5 to about 10 histidine residues may be fused at the N-terminus orthe C-terminus and used as a purification handle in affinitychromatography.

Epitope-tagged Apo-2 can be purified by affinity chromatography usingthe anti-tag antibody. The matrix to which the affinity antibody isattached may include, for instance, agarose, controlled pore glass orpoly(styrenedivinyl)benzene. The epitope-tagged Apo-2 can then be elutedfrom the affinity column using techniques known in the art.

In another embodiment, the chimeric molecule comprises an Apo-2polypeptide fused to an immunoglobulin sequence. The chimeric moleculemay also comprise a particular domain sequence of Apo-2, such as anextracellular domain sequence of Apo-2 fused to an immunoglobulinsequence. This includes chimeras in monomeric, homo- orheteromultimeric, and particularly homo- or heterodimeric, or-tetrameric forms; optionally, the chimeras may be in dimeric forms orhomodimeric heavy chain forms. Generally, these assembledimmunoglobulins will have known unit structures as represented by thefollowing diagrams.

A basic four chain structural unit is the form in which IgG, IgD, andIgE exist. A four chain unit is repeated in the higher molecular weightimmunoglobulins; IgM generally exists as a pentamer of basic four-chainunits held together by disulfide bonds. IgA globulin, and occasionallyIgG globulin, may also exist in a multimeric form in serum. In the caseof multimers, each four chain unit may be the same or different.

The following diagrams depict some exemplary monomer, homo- andheterodimer and homo- and heteromultimer structures. These diagrams aremerely illustrative, and the chains of the multimers are believed to bedisulfide bonded in the same fashion as native immunoglobulins.

In the foregoing diagrams, “A” means an Apo-2 sequence or an Apo-2sequence fused to a heterologous sequence; X is an additional agent,which may be the same as A or different, a portion of an immunoglobulinsuperfamily member such as a variable region or a variable region-likedomain, including a native or chimeric immunoglobulin variable region, atoxin such a pseudomonas exotoxin or ricin, or a sequence functionallybinding to another protein, such as other cytokines (i.e., IL-1,interferon-γ) or cell surface molecules (i.e., NGFR, CD40, OX40, Fasantigen, T2 proteins of Shope and myxoma poxviruses), or a polypeptidetherapeutic agent not otherwise normally associated with a constantdomain; Y is a linker or another receptor sequence; and V_(L), V_(H),C_(L) and C_(H) represent light or heavy chain variable or constantdomains of an immunoglobulin. Structures comprising at least one CRD ofan Apo-2 sequence as “A” and another cell-surface protein having arepetitive pattern of CRDs (such as TNFR) as “X” are specificallyincluded.

It will be understood that the above diagrams are merely exemplary ofthe possible structures of the chimeras of the present invention, and donot encompass all possibilities. For example, there might desirably beseveral different “A”s, “X”s, or “Y”s in any of these constructs. Also,the heavy or light chain constant domains may be originated from thesame or different immunoglobulins. All possible permutations of theillustrated and similar structures are all within the scope of theinvention herein.

In general, the chimeric molecules can be constructed in a fashionsimilar to chimeric antibodies in which a variable domain from anantibody of one species is substituted for the variable domain ofanother species. See, for example, EP 0 125 023; EP 173,494; Munro,Nature, 312:597 (Dec. 13, 1984); Neuberger et al., Nature, 312:604-608(Dec. 13, 1984); Sharon et al., Nature, 309:364-367 (May 24, 1984);Morrison et al., Proc. Nat'l. Acad. Sci. USA, 81:6851-6855 (1984);Morrison et al., Science, 229:1202-1207 (1985); Boulianne et al.,Nature, 312:643-646 (Dec. 13, 1984); Capon et al., Nature, 337:525-531(1989); Traunecker et al., Nature, 339:68-70 (1989).

Alternatively, the chimeric molecules may be constructed as follows. TheDNA including a region encoding the desired sequence, such as an Apo-2and/or TNFR sequence, is cleaved by a restriction enzyme at or proximalto the 3′ end of the DNA encoding the immunoglobulin-like domain(s) andat a point at or near the DNA encoding the N-terminal end of the Apo-2or TNFR polypeptide (where use of a different leader is contemplated) orat or proximal to the N-terminal coding region for TNFR (where thenative signal is employed). This DNA fragment then is readily insertedproximal to DNA encoding an immunoglobulin light or heavy chain constantregion and, if necessary, the resulting construct tailored by deletionalmutagenesis. Preferably, the Ig is a human immunoglobulin when thechimeric molecule is intended for in vivo therapy for humans. DNAencoding immunoglobulin light or heavy chain constant regions is knownor readily available from cDNA Libraries or is synthesized. See forexample, Adams et al., Biochemistry, 19:2711-2719 (1980); Gough et al.,Biochemistry, 19:2702-2710 (1980); Dolby et al., Proc. Natl. Acad. Sci.USA, 77:6027-6031 (1980); Rice et al., Proc. Natl. Acad. Sci.,79:7862-7865 (1982); Falkner et al., Nature, 298:286-288 (1982); andMorrison et al., Ann. Rev. Immunol., 2:239-256 (1984).

Further details of how to prepare such fusions are found in publicationsconcerning the preparation of immunoadhesins. Immunoadhesins in general,and CD4-Ig fusion molecules specifically are disclosed in WO 89/02922,published Apr. 6, 1989. Molecules comprising the extracellular portionof CD4, the receptor for human immunodeficiency virus (HIV), linked toIgG heavy chain constant region are known in the art and have been foundto have a markedly longer half-life and lower clearance than the solubleextracellular portion of CD4 [Capon et al., supra; Byrn et al., Nature,344:667 (1990)]. The construction of specific chimeric TNFR-IgGmolecules is also described in Ashkenazi et al. Proc. Natl. Acad. Sci.,88:10535-10539 (1991); Lesslauer et al. [J. Cell. Biochem. Supplement15F, 1991, p. 115 (P 432)]; and Peppel and Beutler, J. Cell. Biochem.Supplement 15F, 1991, p. 118 (P 439)].

B. Therapeutic and Non-Therapeutic Uses for Apo-2

Apo-2, as disclosed in the present specification, can be employedtherapeutically to induce apoptosis in mammalian cells. This therapy canbe accomplished for instance, using in vivo or ex vivo gene therapytechniques and includes the use of the death domain sequences disclosedherein. The Apo-2 chimeric molecules (including the chimeric moleculescontaining an extracellular domain sequence of Apo-2) comprisingimmunoglobulin sequences can also be employed therapeutically to inhibitapoptosis or NF-KB induction by Apo-2L or by another ligand that Apo-2binds to.

The Apo-2 of the invention also has utility in non-therapeuticapplications. Nucleic acid sequences encoding the Apo-2 may be used as adiagnostic for tissue-specific typing. For example, procedures like insitu hybridization, Northern and Southern blotting, and PCR analysis maybe used to determine whether DNA and/or RNA encoding Apo-2 is present inthe cell type(s) being evaluated. Apo-2 nucleic acid will also be usefulfor the preparation of Apo-2 by the recombinant techniques describedherein.

The isolated Apo-2 may be used in quantitative diagnostic assays as acontrol against which samples containing unknown quantities of Apo-2 maybe prepared. Apo-2 preparations are also useful in generatingantibodies, as standards in assays for Apo-2 (e.g., by labeling Apo-2for use as a standard in a radioimmunoassay, radioreceptor assay, orenzyme-linked immunoassay), in affinity purification techniques, and incompetitive-type receptor binding assays when labeled with, forinstance, radioiodine, enzymes, or fluorophores.

Modified forms of the Apo-2, such as the Apo-2-IgG chimeric molecules(immunoadhesins) described above, can be used as immunogens in producinganti-Apo-2 antibodies.

Nucleic acids which encode Apo-2 or its modified forms can also be usedto generate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding Apo-2 or an appropriate sequence thereof (suchas Apo-2-IgG) can be used to clone genomic DNA encoding Apo-2 inaccordance with established techniques and the genomic sequences used togenerate transgenic animals that contain cells which express DNAencoding Apo-2. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for Apo-2 transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding Apo-2 introduced into the germline of the animal at an embryonic stage can be used to examine theeffect of increased expression of DNA encoding Apo-2. Such animals canbe used as tester animals for reagents thought to confer protectionfrom, for example, pathological conditions associated with excessiveapoptosis. In accordance with this facet of the invention, an animal istreated with the reagent and a reduced incidence of the pathologicalcondition, compared to untreated animals bearing the transgene, wouldindicate a potential therapeutic intervention for the pathologicalcondition. In another embodiment, transgenic animals that carry asoluble form of Apo-2 such as an Apo-2 ECD or an immunoglobulin chimeraof such form could be constructed to test the effect of chronicneutralization of Apo-2L, a ligand of Apo-2.

Alternatively, non-human homologues of Apo-2 can be used to construct anApo-2 “knock out” animal which has a defective or altered gene encodingApo-2 as a result of homologous recombination between the endogenousgene encoding Apo-2 and altered genomic DNA encoding Apo-2 introducedinto an embryonic cell of the animal. For example, cDNA encoding Apo-2can be used to clone genomic DNA encoding Apo-2 in accordance withestablished techniques. A portion of the genomic DNA encoding Apo-2 canbe deleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the Apo-2 polypeptide, including forexample, development of tumors.

C. Anti-Apo-2 Antibody Preparation

The present invention further provides anti-Apo-2 antibodies. Antibodiesagainst Apo-2 may be prepared as follows. Exemplary antibodies includepolyclonal, monoclonal, humanized, bispecific, and heteroconjugateantibodies.

1. Polyclonal Antibodies

The Apo-2 antibodies may comprise polyclonal antibodies. Methods ofpreparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the Apo-2 polypeptide or a fusion proteinthereof. An example of a suitable immunizing agent is an Apo-2-IgGfusion protein, such as an Apo-2 ECD-IgG fusion protein. Cellsexpressing Apo-2 at their surface may also be employed. It may be usefulto conjugate the immunizing agent to a protein known to be immunogenicin the mammal being immunized. Examples of such immunogenic proteinswhich may be employed include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. An aggregating agent such as alum may also be employed toenhance the mammal's immune response. Examples of adjuvants which may beemployed include Freund's complete adjuvant and MPL-TDM adjuvant(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). Theimmunization protocol may be selected by one skilled in the art withoutundue experimentation. The mammal can then be bled, and the serumassayed for antibody titer. If desired, the mammal can be boosted untilthe antibody titer increases or plateaus.

2. Monoclonal Antibodies

The Apo-2 antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, supra. In a hybridoma method, amouse, hamster, or other appropriate host animal, is typically immunized(such as described above) with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The immunizing agent will typically include the Apo-2 polypeptide or afusion protein thereof. An example of a suitable immunizing agent is anApo-2-IgG fusion protein or chimeric molecule. A specific example of anApo-2 ECD-IgG immunogen is described in Example 9 below. Cellsexpressing Apo-2 at their surface may also be employed. Generally,either peripheral blood lymphocytes (“PBLs”) are used if cells of humanorigin are desired, or spleen cells or lymph node cells are used ifnon-human mammalian sources are desired. The lymphocytes are then fusedwith an immortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell [Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental transformed cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed againstApo-2. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

As described in the Examples below, anti-Apo-2 monoclonal antibodieshave been prepared. One of these antibodies, 3F11.39.7, has beendeposited with ATCC and has been assigned deposit accession no.HB-12456. In one embodiment, the monoclonal antibodies of the inventionwill have the same biological characteristics as the monoclonalantibodies secreted by the hybridoma cell line(s) deposited underAccession No. HB-12456. The term “biological characteristics” is used torefer to the in vitro and/or in vivo activities or properties of themonoclonal antibody, such as the ability to specifically bind to Apo-2or to substantially block, induce or enhance Apo-2 activation. Asdisclosed in the present specification, the 3F11.39.7 monoclonalantibody (HB-12456) is characterized as having agonistic activity forinducing apoptosis, binding to the Apo-2 receptor, having blockingactivity as described in the Examples below, and having somecross-reactivity to DR4 but not to DcR1 or DcR2. Optionally, themonoclonal antibody will bind to the same epitope as the 3F11.39.7antibody disclosed herein. This can be determined by conducting variousassays, such as described herein and in the Examples. For instance, todetermine whether a monoclonal antibody has the same specificity as the3F11.39.7 antibody specifically disclosed, one can compare activity inApo-2 blocking and apoptosis induction assays, such as those describedin the Examples below.

The antibodies of the invention may also comprise monovalent antibodies.Methods for preparing monovalent antibodies are well known in the art.For example, one method involves recombinant expression ofimmunoglobulin light chain and modified heavy chain. The heavy chain istruncated generally at any point in the Fc region so as to prevent heavychain crosslinking. Alternatively, the relevant cysteine residues aresubstituted with another amino acid residue or are deleted so as toprevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields an F(ab′)₂ fragment that has two antigencombining sites and is still capable of cross-linking antigen.

The Fab fragments produced in the antibody digestion also contain theconstant domains of the light chain and the first constant domain (CH₁)of the heavy chain. Fab′ fragments differ from Fab fragments by theaddition of a few residues at the carboxy terminus of the heavy chainCH₁ domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)₂antibody fragments originally were produced as pairs of Fab′ fragmentswhich have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known.

3. Humanized Antibodies

The Apo-2 antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino-acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important in order to reduceantigenicity. According to the “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable domain sequences. The human sequencewhich is closest to that of the rodent is then accepted as the humanframework (FR) for the humanized antibody [Sims et al., J. Immunol.,151:2296 (1993); Chothia and Lesk, J. Mol. Biol., 196:901 (1987)].Another method uses a particular framework derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies [Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993)].

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products using threedimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding[see, WO 94/04679 published Mar. 3, 1994].

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. Transfer of thehuman germ-line immunoglobulin gene array in such germ-line mutant micewill result in the production of human antibodies upon antigen challenge[see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno., 7:33 (1993)].

Human antibodies can also be produced in phage display libraries[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1992); Marks et al., J.Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerneret al. are also available for the preparation of human monoclonalantibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95(1991)]. Suitable methods for preparing phage libraries have beenreviewed and are described in Winter et al., Annu. Rev. Immunol.,12:433-55 (1994); Soderlind et al., Immunological Reviews, 130:109-123(1992); Hoogenboom, Tibtech February 1997, Vol. 15; Neri et al., CellBiophysics, 27:47-61 (1995). Libraries of single chain antibodies mayalso be prepared by the methods described in WO 92/01047, WO 92/20791,WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388.Antibody libraries are also commercially available, for example, fromCambridge Antibody Technologies (C.A.T.), Cambridge, UK. Bindingselection against an antigen, in this case Apo-2, can be carried out asdescribed in greater detail in the Examples below.

As described in the Examples below, anti-Apo-2 single-chain Fv (scFv)antibodies have been identified using a phage display library. Three ofthese antibodies, referred to herein as 16E2, 24C4 and 20E6, have beensequenced and characterized. The respective DNA and amino acid sequencesand complementarity determining regions of these antibodies are shown inFIGS. 15A-15C and 16. In one embodiment of the invention, scFv Apo-2antibodies will have the same biological characteristics as the 16E2,24C4 or 20E6 antibodies identified herein. The term “biologicalcharacteristics” is used to refer to the in vitro and/or in vivoactivities or properties of the scFv antibody, such as the ability tospecifically bind to Apo-2 or to substantially induce or enhance Apo-2activation. As disclosed in the present specification, the 16E2, 24C4and 20E6 antibodies are characterized as binding to Apo-2, havingagonistic activity for inducing apoptosis, and having nocross-reactivity to DR4 or several of the other known moleculesrecognized by the Apo-2 ligand. Optionally, the scFv Apo-2 antibody willbind to the same epitope or epitopes recognized by the 16E2, 24C4 or20E6 antibodies disclosed herein. This can be determined by conductingvarious assays, such as described herein and in the Examples. Forinstance, to determine whether a scFv antibody has the same specificityas the 16E2, 24C4 or 20E6 antibodies specifically disclosed, one cancompare activity in apoptosis induction assays, such as those describedin the Examples below.

Optionally the scFv antibodies to Apo-2 may include antibodies whichcontain a VH and VL chain that include one or more complementaritydetermining region (CDR) amino acid sequences identified in FIG. 16 forthe 16E2, 20E6, or 24C4 antibodies.

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe Apo-2, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy-chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1)containing the site necessary for light-chain binding present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy-chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy-chain/light-chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation. This approach is disclosed inWO 94/04690 published Mar. 3, 1994. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology, 121:210 (1986).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

6. Triabodies

Triabodies are also within the scope of the invention. Such antibodiesare described for instance in Iliades et al., FEBS Letters, 409:437-441(1997) and Korrt et al., Protein Engineering, 10:423-433 (1997).

7. Other Modifications

Other modifications of the Apo-2 antibodies are contemplated. Forexample, it may be desirable to modify the antibodies of the inventionwith respect to effector function, so as to enhance the therapeuticeffectiveness of the antibodies. For instance, cysteine residue(s) maybe introduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing [see, e.g., Caron et al., J. Exp. Med.,176:1191-1195 (1992); Shopes, J. Immunol., 148:2918-2922 (1992).Homodimeric antibodies may also be prepared using heterobifunctionalcross-linkers as described in Wolff et al., Cancer Research,53:2560-2565 (1993). Ghetie et al., Proc. Natl. Acad. Sci., 94:7509-7514(1997), further describe preparation of IgG-IgG homodimers and disclosethat such homodimers can enhance apoptotic activity as compared to themonomers. Alternatively, the antibodies can be engineered to have dualFc regions [see, Stevenson et al., Anti-Cancer Drug Design, 3:219-230(1989)].

It may be desirable to modify the amino acid sequences of the antibodiesdisclosed herein. Sequences within the scFv complementary determining orlinker regions (as shown in FIG. 16) may be modified for instance tomodulate the biological activities of these antibodies. Variations inthe full-length scFv sequence or in various domains of the scFvmolecules described herein, can be made, for example, using any of thetechniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding a scFv that results in a change in the amino acidsequence of the scFv as compared with the native sequence scFv.Optionally, the variation is by substitution of at least one amino acidwith any other amino acid in one or more of the domains of the scFvmolecule. The variations can be made using methods known in the art suchas oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al.,Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res.,10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315(1985)], restriction selection mutagenesis [Wells et al., Philos. Trans.R. Soc. London SerA, 317:415 (1986)] or other known techniques can beperformed on the cloned DNA to produce the scFv variant DNA.

The antibodies may optionally be covalently attached or conjugated toone or more chemical groups. A polyol, for example, can be conjugated toan antibody molecule at one or more amino acid residues, includinglysine residues as disclosed in WO 93/00109. Optionally, the polyol is apoly(alkylene glycol), such as poly(ethylene glycol) (PEG), however,those skilled in the art recognize that other polyols, such as, forexample, poly(propylene glycol) and polyethylene-polypropylene glycolcopolymers, can be employed using techniques for conjugating PEG topolypeptides. A variety of methods for pegylating polypeptides have beendescribed. See, e.g. U.S. Pat. No. 4,179,337 which discloses theconjugation of a number of hormones and enzymes to PEG and polypropyleneglycol to produce physiologically active compositions having reducedimmunogenicities.

The antibodies may also be fused or linked to another heterologouspolypeptide or amino acid sequence such as an epitope tag. Epitope tagpolypeptides and methods of their use are described above in Section A,paragraph 8. Any of the tags described herein may be linked to theantibodies. The Examples below, for instance, describe His-tagged andgD-tagged single-chain antibodies.

D. Therapeutic Uses for Apo-2 Antibodies

The Apo-2 antibodies of the invention have therapeutic utility.Agonistic Apo-2 antibodies, for instance, may be employed to activate orstimulate apoptosis in cancer cells. Accordingly, the invention providesmethods for treating cancer using such Apo-2 antibodies. It is of coursecontemplated that the methods of the invention can be employed incombination with still other therapeutic techniques such as surgery.

The agonist is preferably administered to the mammal in a carrier.Suitable carriers and their formulations are described in Remington'sPharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited byOslo et al. Typically, an appropriate amount of apharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of a pharmaceutically-acceptablecarrier include saline, Ringer's solution and dextrose solution. The pHof the solution is preferably from about 5 to about 8, and morepreferably from about 7 to about 7.5. Further carriers include sustainedrelease preparations such as semipermeable matrices of solid hydrophobicpolymers containing the agonist, which matrices are in the form ofshaped articles, e.g., films, liposomes or microparticles. It will beapparent to those persons skilled in the art that certain carriers maybe more preferable depending upon, for instance, the route ofadministration and concentration of agonist being administered.

The agonist antibody can be administered to the mammal by injection(e.g., intravenous, intraperitoneal, subcutaneous, intramuscular), or byother methods such as infusion that ensure its delivery to thebloodstream in an effective form. The agonist may also be administeredby intratumoral, peritumoral, intralesional, or perilesional routes, toexert local as well as systemic therapeutic effects. Local orintravenous injection is preferred.

Effective dosages and schedules for administering the agonist antibodymay be determined empirically, and making such determinations is withinthe skill in the art. Those skilled in the art will understand that thedosage of agonist that must be administered will vary depending on, forexample, the mammal which will receive the agonist, the route ofadministration, the particular type of agonist used and other drugsbeing administered to the mammal. Guidance in selecting appropriatedoses for antibody agonists is found in the literature on therapeuticuses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone etal., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haberet al., eds., Raven Press, New York (1977) pp. 365-389. A typical dailydosage of the agonist used alone might range from about 1 μg/kg to up to100 mg/kg of body weight or more per day, depending on the factorsmentioned above.

The agonist antibody may also be administered to the mammal incombination with effective amounts of one or more other therapeuticagents or in conjunction with radiation treatment. Therapeutic agentscontemplated include chemotherapeutics as well as immunoadjuvants andcytokines. Chemotherapies contemplated by the invention include chemicalsubstances or drugs which are known in the art and are commerciallyavailable, such as Doxorubicin, 5-Fluorouracil, Cytosine arabinoside(“Ara-C”), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol,Methotrexate, Cisplatin, Melphalan, Vinblastine and Carboplatin. Theagonist may be administered sequentially or concurrently with the one ormore other therapeutic agents. The amounts of agonist and therapeuticagent depend, for example, on what type of drugs are used, the cancerbeing treated, and the scheduling and routes of administration but wouldgenerally be less than if each were used individually.

Following administration of agonist to the mammal, the mammal's cancerand physiological condition can be monitored in various ways well knownto the skilled practitioner. For instance, tumor mass may be observedphysically or by standard x-ray imaging techniques.

The Apo-2 antibodies of the invention may also be useful in enhancingimmune-mediated cell death in cells expressing Apo-2, for instance,through complement fixation or ADCC. Alternatively, antagonisticantibodies may be used to block excessive apoptosis (for instance inneurodegenerative disease) or to block potential autoimmune/inflammatoryeffects of Apo-2 resulting from NF-κB activation. Such antagonisticantibodies can be utilized according to the therapeutic methods andtechniques described above.

E. Non-Therapeutic Uses for Apo-2 Antibodies

Apo-2 antibodies may further be used in diagnostic assays for Apo-2,e.g., detecting its expression in specific cells, tissues, or serum.Various diagnostic assay techniques known in the art may be used, suchas competitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Apo-2 antibodies also are useful for the affinity purification of Apo-2from recombinant cell culture or natural sources. In this process, theantibodies against Apo-2 are immobilized on a suitable support, such asSephadex resin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing theApo-2 to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the Apo-2, which is bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the Apo-2 from the antibody.

F. Kits Containing Apo-2 or Apo-2 Antibodies

In a further embodiment of the invention, there are provided articles ofmanufacture and kits containing Apo-2 or Apo-2 antibodies which can beused, for instance, for the therapeutic or non-therapeutic applicationsdescribed above. The article of manufacture comprises a container with alabel. Suitable containers include, for example, bottles, vials, andtest tubes. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition whichincludes an active agent that is effective for therapeutic ornon-therapeutic applications, such as described above. The active agentin the composition is Apo-2 or an Apo-2 antibody. The label on thecontainer indicates that the composition is used for a specific therapyor non-therapeutic application, and may also indicate directions foreither in vivo or in vitro use, such as those described above.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

All restriction enzymes referred to in the examples were purchased fromNew England Biolabs and used according to manufacturer's instructions.All other commercially available reagents referred to in the exampleswere used according to manufacturer's instructions unless otherwiseindicated. The source of those cells identified in the followingexamples, and throughout the specification, by ATCC accession numbers isthe American Type Culture Collection, Manassas, Va.

Example 1

Isolation of cDNA Clones Encoding Human Apo-2

Expressed sequence tag (EST) DNA databases (LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.) were searched and an EST wasidentified which showed homology to the death domain of the Apo-3receptor [Marsters et al., Curr. Biol., 6:750 (1996)]. Human pancreasand kidney lgt10 bacteriophage cDNA libraries (both purchased fromClontech) were ligated into pRK5 vectors as follows. Reagents were addedtogether and incubated at 16° C. for 16 hours: 5×T4 ligase buffer (3ml); pRK5, XhoI, NotI digested vector, 0.5 mg, 1 ml); cDNA (5 ml) anddistilled water (6 ml). Subsequently, additional distilled water (70 ml)and 10 mg/ml tRNA (0.1 ml) were added and the entire reaction wasextracted through phenol:chloroform:isoamyl alcohol (25:24:1). Theaqueous phase was removed, collected and diluted into 5M NaCl (10 ml)and absolute ethanol (−20° C., 250 ml). This was then centrifuged for 20minutes at 14,000×g, decanted, and the pellet resuspended into 70%ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000×g. TheDNA pellet was then dried in a speedvac and eluted into distilled water(3 ml) for use in the subsequent procedure.

The ligated cDNA/pRK5 vector DNA prepared previously was chilled on iceto which was added electrocompetent DH10B bacteria (Life Tech., 20 ml).The bacteria vector mixture was then electroporated as per themanufacturers recommendation. Subsequently SOC media (1 ml) was addedand the mixture was incubated at 37° C. for 30 minutes. Thetransformants were then plated onto 20 standard 150 mm LB platescontaining ampicillin and incubated for 16 hours (37° C.) to allow thecolonies to grow. Positive colonies were then scraped off and the DNAisolated from the bacterial pellet using standard CsCl-gradientprotocols.

An enriched 5′-cDNA library was then constructed to obtain a bias ofcDNA fragments which preferentially represents the 5′ ends of cDNA'scontained within the library. 10 mg of the pooled isolated full-lengthlibrary plasmid DNA (41 ml) was combined with Not 1 restriction buffer(New England Biolabs, 5 ml) and Not 1 (New England Biolabs, 4 ml) andincubated at 37° C. for one hour. The reaction was extracted throughphenol:chloroform:isoamyl alcohol (25:24:1, 50 ml), the aqueous phaseremoved, collected and resuspended into 5M NaCl (5 ml) and absoluteethanol (−20° C., 150 ml). This was then centrifuged for 20 minutes at14,000×g, decanted, resuspended into 70% ethanol (0.5 ml) andcentrifuged again for 2 minutes at 14,000×g. The supernatant was thenremoved, the pellet dried in a speedvac and resuspended in distilledwater (10 ml).

The following reagents were brought together and incubated at 37° C. for2 hours: distilled water (3 ml); linearized DNA library (1 mg, 1 ml);Ribonucleotide mix (Invitrogen, 10 ml); transcription buffer(Invitrogen, 2 ml) and Sp6 enzyme mix. The reaction was then extractedthrough phenol:chloroform:isoamyl alcohol (25:24:1, 50 ml) and theaqueous phase was removed, collected and resuspended into 5M NaCl (5 ml)and absolute ethanol (−20° C., 150 ml) and centrifuged for 20 minutes at14,000×g. The pellet was then decanted and resuspended in 70% ethanol(0.5 ml), centrifuged again for 2 minutes at 14,000×g, decanted, driedin a speedvac and resuspended into distilled water (10 ml).

The following reagents were added together and incubated at 16° C. for16 hours: 5×T4 ligase buffer (Life Tech., 3 ml); pRK5 Cla-Sal digestedvector, 0.5 mg, 1 ml); cDNA (5 ml); distilled water (6 ml).Subsequently, additional distilled water (70 ml) and 10 mg/ml tRNA (0.1ml) was added and the entire reaction was extracted throughphenol:chloroform:isoamyl alcohol (25:24:1, 100 ml). The aqueous phasewas removed, collected and diluted by 5M NaCl (10 ml) and absoluteethanol (−20° C., 250 ml) and centrifuged for 20 minutes at 14,000×g.The DNA pellet was decanted, resuspended into 70% ethanol (0.5 ml) andcentrifuged again for 2 minutes at 14,000×g. The supernatant was removedand the residue pellet was dried in a speedvac and resuspended indistilled water (3 ml). The ligated cDNA/pSST-amy.1 vector DNA waschilled on ice to which was added electrocompetent DH10B bacteria (LifeTech., 20 ml). The bacteria vector mixture was then electroporated asrecommended by the manufacturer. Subsequently, SOC media (Life Tech., 1ml) was added and the mixture was incubated at 37° C. for 30 minutes.The transformants were then plated onto 20 standard 150 mm LB platescontaining ampicillin and incubated for 16 hours (37° C.). Positivecolonies were scraped off the plates and the DNA was isolated from thebacterial pellet using standard protocols, e.g. CsCl-gradient.

The cDNA libraries were screened by hybridization with a syntheticoligonucleotide probe:GGGAGCCGCTCATGAGGAAGTTGGGCCTCATGGACAATGAGATAAAGGTGGCTAAAGCTGAGGCAGCGGG(SEQ ID NO:3) based on the EST.

Three cDNA clones were sequenced in entirety. The overlapping codingregions of the cDNAs were identical except for codon 410 (using thenumbering system for FIG. 1); this position encoded a leucine residue(TTG) in both pancreatic cDNAs, and a methionine residue (ATG) in thekidney cDNA, possibly due to polymorphism.

The entire nucleotide sequence of Apo-2 is shown in FIG. 1 (SEQ IDNO:2). Clone 27868 (also referred to as pRK5-Apo-2 deposited as ATCC209021, as indicated below) contains a single open reading frame with anapparent translational initiation site at nucleotide positions 140-142[Kozak et al., supra] and ending at the stop codon found at nucleotidepositions 1373-1375 (FIG. 1; SEQ ID NO:2). The predicted polypeptideprecursor is 411 amino acids long, a type I transmembrane protein, andhas a calculated molecular weight of approximately 45 kDa. Hydropathyanalysis (not shown) suggested the presence of a signal sequence(residues 1-53), followed by an extracellular domain (residues 54-182),a transmembrane domain (residues 183-208), and an intracellular domain(residues 209-411) (FIG. 2A; SEQ ID NO:1). N-terminal amino acidsequence analysis of Apo-2-IgG expressed in 293 cells showed that themature polypeptide starts at amino acid residue 54, indicating that theactual signal sequence comprises residues 1-53. Apo-2 polypeptide isobtained or obtainable by expressing the molecule encoded by the cDNAinsert of the deposited ATCC 209021 vector.

TNF receptor family proteins are typically characterized by the presenceof multiple (usually four) cysteine-rich domains in their extracellularregions—each cysteine-rich domain being approximately 45 amino acidslong and containing approximately 6, regularly spaced, cysteineresidues. Based on the crystal structure of the type 1 TNF receptor, thecysteines in each domain typically form three disulfide bonds in whichusually cysteines 1 and 2, 3 and 5, and 4 and 6 are paired together.Like DR4, Apo-2 contains two extracellular cysteine-rich pseudorepeats(FIG. 2A), whereas other identified mammalian TNFR family memberscontain three or more such domains [Smith et al., Cell, 76:959 (1994)].

The cytoplasmic region of Apo-2 contains a death domain (amino acidresidues 324-391 shown in FIG. 1; see also FIG. 2A) which showssignificantly more amino acid sequence identity to the death domain ofDR4 (64%) than to the death domain of TNFR1 (30%); CD95 (19%); orApo-3/DR3 (29%) (FIG. 2B). Four out of six death domain amino acids thatare required for signaling by TNFR1 [Tartaglia et al., supra] areconserved in Apo-2 while the other two residues are semi-conserved (seeFIG. 2B).

Based on an alignment analysis (using the ALIGN™ computer program) ofthe full-length sequence, Apo-2 shows more sequence identity to DR4(55%) than to other apoptosis-linked receptors, such as TNFR1 (19%);CD95 (17%); or Apo-3 (also referred to as DR3, WSL-1 or TRAMP) (29%).

Example 2

A. Expression of Apo-2 ECD

A soluble extracellular domain (ECD) fusion construct was prepared. AnApo-2 ECD (amino acid residues 1-184 shown in FIG. 1) was obtained byPCR and fused to a C-terminal Flag epitope tag (Sigma). (The Apo-2 ECDconstruct included residues 183 and 184 shown in FIG. 1 to provideflexibility at the junction, even though residues 183 and 184 arepredicted to be in the transmembrane region). The Flag epitope-taggedmolecule was then inserted into pRK5, and expressed by transienttransfection into human 293 cells (ATCC CRL 1573).

After a 48 hour incubation, the cell supernatants were collected andeither used directly for co-precipitation studies (see Example 3) orsubjected to purification of the Apo-2 ECD-Flag by affinitychromatography on anti-Flag agarose beads, according to manufacturer'sinstructions (Sigma).

B. Expression of Apo-2 ECD as an Immunoadhesin

A soluble Apo-2 ECD immunoadhesin construct was prepared. The Apo-2 ECD(amino acids 1-184 shown in FIG. 1) was fused to the hinge and Fc regionof human immunoglobulin G₁ heavy chain in pRK5 as described previously[Ashkenazi et al., Proc. Natl. Acad. Sci., 88:10535-10539 (1991)]. Theimmunoadhesin was expressed by transient transfection into human 293cells and purified from cell supernatants by protein A affinitychromatography, as described by Ashkenazi et al., supra.

Example 3

Immunoprecipitation Assay Showing Binding Interaction Between Apo-2 andApo-2 Ligand

To determine whether Apo-2 and Apo-2L interact or associate with eachother, supernatants from mock-transfected 293 cells or from 293 cellstransfected with Apo-2 ECD-Flag (described in Example 2 above) (5 ml)were incubated with 5 μg poly-histidine-tagged soluble Apo-2L [Pitti etal., supra] for 30 minutes at room temperature and then analyzed forcomplex formation by a co-precipitation assay.

The samples were subjected to immunoprecipitation using 25 μl anti-Flagconjugated agarose beads (Sigma) or Nickel-conjugated agarose beads(Qiagen). After a 1.5 hour incubation at 4° C., the beads were spun downand washed four times in phosphate buffered saline (PBS). By usinganti-Flag agarose, the Apo-2L was precipitated through the Flag-taggedApo-2 ECD; by using Nickel-agarose, the Apo-2 ECD was precipitatedthrough the His-tagged Apo-2L. The precipitated proteins were releasedby boiling the beads for 5 minutes in SDS-PAGE buffer, resolved byelectrophoresis on 12% polyacrylamide gels, and then detected byimmunoblot with anti-Apo-2L or anti-Flag antibody (2 μg/ml) as describedin Marsters et al., J. Biol. Chem., (1997).

The results, shown in FIG. 3, indicate that the Apo-2 ECD and Apo-2L canassociate with each other.

The binding interaction was further analyzed by purifying Apo-2 ECD fromthe transfected 293 cell supernatants with anti-Flag beads (see Example2) and then analyzing the samples on a BIACORE™ instrument. The BIACORE™analysis indicated a dissociation constant (K_(d)) of about 1 nM.BIACORE™ analysis also showed that the Apo-2 ECD is not capable ofbinding other apoptosis-inducing TNF family members, namely, TNF-alpha(Genentech, Inc., Pennica et al., Nature, 312:712 (1984),lymphotoxin-alpha (Genentech, Inc.), or Fas/Apo-1 ligand (AlexisBiochemicals). The data thus shows that Apo-2 is a specific receptor forApo-2L.

Example 4

Induction of Apoptosis by Apo-2

Because death domains can function as oligomerization interfaces,over-expression of receptors that contain death domains may lead toactivation of signaling in the absence of ligand [Frazer et al., supra,Nagata et al., supra]. To determine whether Apo-2 was capable ofinducing cell death, human 293 cells or HeLa cells (ATCC CCL 2.2) weretransiently transfected by calcium phosphate precipitation (293 cells)or electroporation (HeLa cells) with a pRK5 vector or pRK5-basedplasmids encoding Apo-2 and/or CrmA. When applicable, the total amountof plasmid DNA was adjusted by adding vector DNA. Apoptosis was assessed24 hours after transfection by morphology (FIG. 4A); DNA fragmentation(FIG. 4B); or by FACS analysis of phosphatydilserine exposure (FIG. 4C)as described in Marsters et al., Curr. Biol., 6: 1669 (1996). As shownin FIGS. 4A and 4B, the Apo-2 transfected 293 cells underwent markedapoptosis.

For samples assayed by FACS, the HeLa cells were co-transfected withpRK5-CD4 as a marker for transfection and apoptosis was determined inCD4-expressing cells; FADD was co-transfected with the Apo-2 plasmid;the data are means±SEM of at least three experiments, as described inMarsters et al., Curr. Biol., 6:1669 (1996). The caspase inhibitors,DEVD-fmk (Enzyme Systems) or z-VAD-fmk (Research Biochemicals Intl.)were added at 200 μM at the time of transfection. As shown in FIG. 4C,the caspase inhibitors CrmA, DEVD-fmk, and z-VAD-fmk blocked apoptosisinduction by Apo-2, indicating the involvement of Ced-3-like proteasesin this response.

FADD is an adaptor protein that mediates apoptosis activation by CD95,TNFR1, and Apo-3/DR3 [Nagata et al., supra], but does not appearnecessary for apoptosis induction by Apo-2L [Marsters et al., supra] orby DR4 [Pan et al., supra]. A dominant-negative mutant form of FADD,which blocks apoptosis induction by CD95, TNFR1, or Apo-3/DR3 [Frazer etal., supra; Nagata et al., supra; Chinnayian et al., supra] did notinhibit apoptosis induction by Apo-2 when co-transfected into HeLa cellswith Apo-2 (FIG. 4C). These results suggest that Apo-2 signals apoptosisindependently of FADD. Consistent with this conclusion, aglutathione-S-transferase fusion protein containing the Apo-2cytoplasmic region did not bind to in vitro transcribed and translatedFADD (data not shown).

Example 5

Inhibition of Apo-2L Activity by Soluble Apo-2 ECD

Soluble Apo-2L (0.5 μg/ml, prepared as described in Pitti et al., supra)was pre-incubated for 1 hour at room temperature with PBS buffer oraffinity-purified Apo-2 ECD (5 μg/ml) together with anti-Flag antibody(Sigma) (1 μg/ml) and added to HeLa cells. After a 5 hour incubation,the cells were analyzed for apoptosis by FACS (as above) (FIG. 4D).

Apo-2L induced marked apoptosis in HeLa cells, and the soluble Apo-2 ECDwas capable of blocking Apo-2L action (FIG. 4D), confirming a specificinteraction between Apo-2L and Apo-2. Similar results were obtained withthe Apo-2 ECD immunoadhesin (FIG. 4D). Dose-response analysis showedhalf-maximal inhibition at approximately 0.3 nM Apo-2 immunoadhesin(FIG. 4E).

Example 6

Activation of NF-κB by Apo-2

An assay was conducted to determine whether Apo-2 activates NF-κB.

HeLa cells were transfected with pRK5 expression plasmids encodingfull-length native sequence Apo-2, DR4 or Apo-3 and harvested 24 hoursafter transfection. Nuclear extracts were prepared and 1 μg of nuclearprotein was reacted with a ³²P-labelled NF-κB-specific syntheticoligonucleotide probe ATCAGGGACTTTCCGCTGGGGACTTTCCG (SEQ ID NO:4) [see,also, MacKay et al., J. Immunol., 153:5274-5284 (1994)], alone ortogether with a 50-fold excess of unlabelled probe, or with anirrelevant ³²P-labelled synthetic oligonucleotideAGGATGGGAAGTGTGTGATATATCCTTGAT (SEQ ID NO:5). In some samples, antibodyto p65/RelA subunits of NF-κB (1 μg/ml; Santa Cruz Biotechnology) wasadded. DNA binding was analyzed by an electrophoretic mobility shiftassay as described by Hsu et al., supra; Marsters et al., supra, andMacKay et al., supra.

The results are shown in FIG. 5. As shown in FIG. 5A, upon transfectioninto HeLa cells, both Apo-2 and DR4 induced significant NF-κB activationas measured by the electrophoretic mobility shift assay; the level ofactivation was comparable to activation observed for Apo-3/DR3. Antibodyto the p65/RelA subunit of NF-κB inhibited the mobility of the NF-κBprobe, implicating p65 in the response to all 3 receptors.

An assay was also conducted to determine if Apo-2L itself can regulateNF-κB activity. HeLa cells or MCF7 cells (human breast adenocarcinomacell line, ATCC HTB 22) were treated with PBS buffer, soluble Apo-2L(Pitti et al., supra) or TNF-alpha (Genentech, Inc., see Pennica et al.,Nature, 312:721 (1984)) (1 μg/ml) and assayed for NF-κB activity asabove. The results are shown in FIG. 5B. The Apo-2L induced asignificant NF-κB activation in the treated HeLa cells but not in thetreated MCF7 cells; the TNF-alpha induced a more pronounced activationin both cell lines. Several studies have disclosed that NF-κB activationby TNF can protect cells against TNF-induced apoptosis [Nagata, supra].

The effects of a NF-κB inhibitor, ALLN (N-acetyl-Leu-Leu-norleucinal)and a transcription inhibitor, cyclohexamide, were also tested. The HeLacells (plated in 6-well dishes) were preincubated with PBS buffer, ALLN(Calbiochem) (40 μg/ml) or cyclohexamide (Sigma) (50 μg/ml) for 1 hourbefore addition of Apo-2L (1 μg/ml). After a 5 hour incubation,apoptosis was analyzed by FACS (see FIG. 5C).

The results are shown in FIG. 5C. Both ALLN and cyclohexamide increasedthe level of Apo-2L-induced apoptosis in the HeLa cells. The dataindicates that Apo-2L can induce protective NF-κB-dependent genes. Thedata also indicates that Apo-2L is capable of activating NF-κB incertain cell lines and that both Apo-2 and DR4 may mediate thatfunction.

Example 7

Expression of Apo-2 in Mammalian Tissues

A. Northern Blot Analysis

Expression of Apo-2 mRNA in human tissues was examined by Northern blotanalysis. Human RNA blots were hybridized to a 4.6 kilobase ³²P-labelledDNA probe based on the full length Apo-2 cDNA; the probe was generatedby digesting the pRK5-Apo-2 plasmid with EcoRI. Human fetal RNA blot MTN(Clontech), human adult RNA blot MTN-II (Clontech), and human cancercell line RNA blot (Clontech) were incubated with the DNA probes. Blotswere incubated with the probes in hybridization buffer (5×SSPE;2×Denhardt's solution; 100 mg/mL denatured sheared salmon sperm DNA; 50%formamide; 2% SDS) for 60 hours at 42° C. The blots were washed severaltimes in 2×SSC; 0.05% SDS for 1 hour at room temperature, followed by a30 minute wash in 0.1×SSC; 0.1% SDS at 50° C. The blots were developedafter overnight exposure.

As shown in FIG. 6A, a predominant mRNA transcript of approximately 4.6kb was detected in multiple tissues. Expression was relatively high infetal and adult liver and lung, and in adult ovary and peripheral bloodleukocytes (PBL), while no mRNA expression was detected in fetal andadult brain. Intermediate levels of expression were seen in adult colon,small intestine, testis, prostate, thymus, pancreas, kidney, skeletalmuscle, placenta, and heart. Several adult tissues that express Apo-2,e.g., PBL, ovary, and spleen, have been shown previously to express DR4[Pan et al., supra], however, the relative levels of expression of eachreceptor mRNA appear to be different.

As shown in FIG. 6B, Apo-2 mRNA was expressed relatively high in 6 of 8human cancer cell lines examined, namely, HL60 promyelocytic leukemia,HeLa S3 cervical carcinoma, K562 chronic myelogenous leukemia, SW 480colorectal adenocarcinoma, A549 lung carcinoma, and G361 melanoma. Therewas also detectable expression in Burkitt's lymphoma (Raji) cells. Thus,Apo-2 may be useful as a target for inducing apoptosis in cancer cellsfrom lymphoid as well as non-lymphoid tumors.

B. In Situ Hybridization

Expression of Apo-2 in normal and in cancerous human tissues wasexamined by in situ hybridization. In addition, several different chimpand rhesus monkey tissues were examined for Apo-2 expression. Thesetissues included: human fetal tissues (E12-E16 weeks)—placenta,umbilical cord, liver, kidney, adrenal gland, thyroid, lung, heart,great vessels, esophagus, stomach, small intestine, spleen, thymus,pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb;adult human tissues—kidney, bladder, adrenal gland, spleen, lymph node,pancreas, lung, skin, retina, liver; chimp tissues—salivary gland,stomach, thyroid, parathyroid, tongue, thymus, ovary, lymph node, andperipheral nerve; rhesus monkey tissues—cerebral cortex, hippocampus,cerebellum and penis; human tumor tissue—lung adenocarcinoma, testis,lung carcinoma, breast carcinoma, fibroadenoma, soft tissue sarcoma.

Tissue samples were paraffin-embedded and sectioned. Later, thesectioned tissues were deparaffinized and the slides placed in water.The slides were rinsed twice for five minutes at room temperature in2×SSC. After rinsing, the slides were placed in 20 μg/ml proteinase K(in Rnase-free buffer) for 15 minutes at 37° C. (for fetal tissues) or8× proteinase K for 30 minutes at 37° C. (for formalin tissues). Theslides were then rinsed again in 0.5×SSC and dehydrated. Prior tohybridization, the slides were placed in a plastic box lined with buffer(4×SSC, 50% formamide)—saturated filter paper. The tissues were coveredwith 50 μl hybridization buffer (3.75 g Dextran sulfate plus 6 ml water;vortexed and heated for 2 minutes; cooled on ice and 18.75 ml formamide,3.75 ml 20×SSC and 9 ml water added) and incubated at 42° C. for 1 to 4hours.

Hybridization was conducted using a ³³P labelled probe consisting ofnucleotides 706-1259 of SEQ ID NO:2. The probe was added to the slidesin hybridization buffer and incubated overnight at 55° C. Multiplewashing steps were then performed sequentially as follows: twice for 10minutes at room temperature in 2×SSC, EDTA buffer (400 ml 20×SSC, 16 ml0.25M EDTA); once for 30 minutes at 37° C. in 20 μg/ml RNase A; twicefor 10 minutes at room temperature in 2×SSC, EDTA buffer; once for 2hours at 55° C. in 0.1×SSC, EDTA buffer; twice for 10 minutes at roomtemperature in 0.5×SSC. Dehydration was performed for 2 minutes each in50%, 70%, 90% EtOH containing 0.3 M NH₄AC. Finally, the slides wereair-dried for 2 hours and exposed to film.

Expression of Apo-2 in the fetal tissues appeared strongest overhepatocytes in liver, developing glomeruli in kidney, adrenal cortex,and epithelium of gastrointestinal tract. Moderate expression wasobserved over epithelial cells in lung and at sites of vascularizationof a bone growth plate. A relatively low level expression was observedover thyroid epithelial cells and cells in cardiac ventricles.Expression was observed over lymphoid cells in the thymic medulla,developing lymph glands and placenta cytotrophoblast cells.

Expression of Apo-2 in adult tissues was observed over resting oocytesin primordial follicles and low levels over granulosa cells ofdeveloping follicles in chimp ovary. Expression was observed incirrhotic livers over hepatocytes at the edge of nodules (i.e., area ofdamage, normal adult liver was negative). Other tissues were negativefor expression.

In the cancer tissues examined, Apo-2 expression was found in two lungadenocarcinomas and two germ cell tumors of the testis. Two additionallung carcinomas (one squamous) were negative. One of five breastcarcinomas was positive (there was expression in normal breast tissue).In a fibroadenoma, there appeared to be expression over both epithelialand stromal elements. A soft tissue sarcoma was also positive. Othertissues examined were negative.

Example 8

Chromosomal Localization of the Apo-2 Gene

Chromosomal localization of the human Apo-2 gene was examined byradiation hybrid (RH) panel analysis. RH mapping was performed by PCRusing a human-mouse cell radiation hybrid panel (Research Genetics) andprimers based on the coding region of the Apo-2 cDNA [Gelb et al., Hum.Genet., 98:141 (1996)]. Analysis of the PCR data using the StanfordHuman Genome Center Database indicates that Apo-2 is linked to themarker D8S481, with an LOD of 11.05; D8S481 is linked in turn toD8S2055, which maps to human chromosome 8p21. A similar analysis of DR4showed that DR4 is linked to the marker D8S2127 (with an LOD of 13.00),which maps also to human chromosome 8p21.

To Applicants' present knowledge, to date, no other member of the TNFRgene family has been located to chromosome 8.

Example 9

Preparation of Monoclonal Antibodies Specific for Apo-2

Balb/c mice (obtained from Charles River Laboratories) were immunized byinjecting 0.5 μg/50 μl of an Apo-2 ECD immunoadhesin protein (diluted inMPL-TDM adjuvant purchased from Ribi Immunochemical Research Inc.,Hamilton, Mont.) 11 times into each hind foot pad at 3-4 day intervals.The Apo-2 ECD immunoadhesin protein was generated by fusing anextracellular domain sequence of Apo-2 (amino acids 1-184 shown inFIG. 1) to the hinge and Fc region of human immunoglobulin G₁ heavychain in pRK5 as described previously [Ashkenazi et al., Proc. Natl.Acad. Sci., 88:10535-10539 (1991)]. The immunoadhesin protein wasexpressed by transient transfection into human 293 cells and purifiedfrom cell supernatants by protein A affinity chromatography, asdescribed by Ashkenazi et al., supra (See also Example 2B above).

Three days after the final boost, popliteal lymph nodes were removedfrom the mice and a single cell suspension was prepared in DMEM media(obtained from Biowhitakker Corp.) supplemented with 1%penicillin-streptomycin. The lymph node cells were then fused withmurine myeloma cells P3X63AgU.1 (ATCC CRL 1597) using 35% polyethyleneglycol and cultured in 96-well culture plates. Hybridomas resulting fromthe fusion were selected in HAT medium. Ten days after the fusion,hybridoma culture supernatants were screened in an ELISA to test for thepresence of monoclonal antibodies binding to the Apo-2 ECD immunoadhesinprotein.

In the ELISA, 96-well microtiter plates (Maxisorb; Nunc, Kamstrup,Denmark) were coated by adding 50 μl of 2 μg/ml goat anti-human IgG Fc(purchased from Cappel Laboratories) in PBS to each well and incubatingat 4° C. overnight. The plates were then washed three times with washbuffer (PBS containing 0.05% Tween 20). The wells in the microtiterplates were then blocked with 50 μl of 2.0% bovine serum albumin in PBSand incubated at room temperature for 1 hour. The plates were thenwashed again three times with wash buffer.

After the washing step, 50 μl of 0.4 μg/ml Apo-2 ECD immunoadhesinprotein (as described above) in assay buffer was added to each well. Theplates were incubated for 1 hour at room temperature on a shakerapparatus, followed by washing three times with wash buffer.

Following the wash steps, 100 μl of the hybridoma supernatants orpurified antibody (using Protein A-sepharose columns) (1 μg/ml) wasadded to designated wells in the presence of CD4-IgG. 100 μl ofP3X63AgU.1 myeloma cell conditioned medium was added to other designatedwells as controls. The plates were incubated at room temperature for 1hour on a shaker apparatus and then washed three times with wash buffer.

Next, 50 μl HRP-conjugated goat anti-mouse IgG Fc (purchased from CappelLaboratories), diluted 1:1000 in assay buffer (0.5% bovine serumalbumin, 0.05% Tween-20, 0.01% Thimersol in PBS), was added to each welland the plates incubated for 1 hour at room temperature on a shakerapparatus. The plates were washed three times with wash buffer, followedby addition of 50 μl of substrate (TMB microwell peroxidase substrate,Kirkegaard & Perry, Gaithersburg, Md.) to each well and incubation atroom temperature for 10 minutes. The reaction was stopped by adding 50μl of TMB 1-component stop solution (diethyl glycol, Kirkegaard & Perry)to each well, and absorbance at 450 nm was read in an automatedmicrotiter plate reader.

Of the hybridoma supernatants screened in the ELISA, 22 supernatantstested positive (calculated as approximately 4 times above background).The supernatants testing positive in the ELISA were further analyzed byFACS analysis using 9D cells (a human B lymphoid cell line expressingApo-2; Genentech, Inc.) and FITC-conjugated goat anti-mouse IgG. Forthis analysis, 25 μl of cells suspended (at 4×10⁶ cells/ml) in cellsorter buffer (PBS containing 1% FCS and 0.02% NaN₃) were added toU-bottom microtiter wells, mixed with 100 μl of culture supernatant orpurified antibody (purified on Protein A-sepharose columns) (10 μg/ml)in cell sorter buffer, and incubated for 30 minutes on ice. The cellswere then washed and incubated with 100 μl FITC-conjugated goatanti-mouse IgG for 30 minutes at 4° C. Cells were then washed twice,resuspended in 150 μl of cell sorter buffer and then analyzed by FACScan(Becton Dickinson, Mountain View, Calif.). FACS analysis showed 8/22supernatants were positive for anti-Apo-2 antibodies.

FIG. 7 shows the FACS staining of 9D cells incubated with one of theApo-2 antibodies, referred to as 3F11.39.7. As shown in FIG. 7, the3F11.39.7 antibody recognizes the Apo-2 receptor expressed in 9D cells.

Example 10

Assay for Ability of Apo-2 Abs to Agonistically Induce Apoptosis

Hybridoma supernatants and purified antibodies (as described in Example9 above) were tested for activity to induce Apo-2 mediated 9D cellapoptosis. The 9D cells (5×10⁵ cells/0.1 ml) were incubated with varyingconcentrations of antibodies in 100 μl complete RPMI media at 4° C. for15 minutes. The cells were then incubated for 5 minutes at 37° C. and 10μg of goat anti-mouse IgG Fc antibody (Cappel Laboratories) in 300 μl ofcomplete RPMI was added to some of the cell samples. At this point, thecells were incubated overnight at 37° C. and in the presence of 7% CO₂.The cells were then harvested and washed once with PBS. The viability ofthe cells was determined by staining of FITC-annexin V binding tophosphatidylserine according to manufacturer recommendations (Clontech).The cells were washed in PBS and resuspended in 200 μl binding buffer.Ten μl of annexin-V-FITC (1 μg/ml) and 10 μl of propidium iodide wereadded to the cells. After incubation for 15 minutes in the dark, the 9Dcells were analyzed by FACS.

As shown in FIG. 8, the 3F11.39.7 antibody (in the absence of the goatanti-mouse TgG Fc) induced apoptosis in the 9D cells as compared to thecontrol antibodies. Agonistic activity, however, was enhanced by Apo-2receptor cross-linking in the presence of the goat anti-mouse IgG Fc(see FIG. 9). This enhanced apoptosis (FIG. 9) by the combination ofantibodies is comparable to the apoptotic activity of Apo-2L in 9D cells(data not shown).

Example 11

Assay for Antibody Ability to Block Apo-2 Ligand-Induced Apoptosis

Hybridoma supernatants and purified antibodies (as described in Example9 above) were tested for activity to block Apo-2 ligand induced 9D cellapoptosis. The 9D cells (5×10⁵ cells/0.1 ml) were suspended in completeRPMI media (RPMI plus 10% FCS, glutamine, nonessential amino acids,penicillin, streptomycin, sodium pyruvate) and placed into individualFalcon 2052 tubes. Cells were then incubated with 10 μg of antibodies in200 μl media for 15 minutes on ice. 0.2 ml of Apo-2 ligand (2.5 μg/ml)(soluble His-tagged Apo-2L prepared as described in WO 97/25428; seealso Pitti et al., supra) was suspended into complete RPMI media, andthen added into the tubes containing the 9D cells. The 9D cells wereincubated overnight at 37° C. and in the presence of 7% CO₂ Theincubated cells were then harvested and washed once with PBS. Theviability of the cells was determined by staining of FITC-annexin Vbinding to phosphatidylserine according to manufacturer recommendations(Clontech). Specifically, the cells were washed in PBS and resuspendedin 200 μl binding buffer. Ten μl of annexin-V-FITC (1 μg/ml) and 10 μlof propidium iodide were added to the cells. After incubation for 15minutes in the dark, the 9D cells were analyzed by FACS.

The results are shown in FIG. 10. Since 9D cells 2b express more thanone receptor for Apo-2L, Apo-2L can induce apoptosis in the 9D cells byinteracting with either Apo-2 or the DR4 receptor. Thus, to detect anyblocking activity of the Apo-2 antibodies, the interaction between DR4and Apo-2L needed to be blocked. In combination with the anti-DR4antibody, 4H6.17.8 (ATCC HB-12455), the Apo-2 antibody 3F11.39.7 wasable to block approximately 50% of apoptosis induced by Apo-2L. Theremaining approximately 50% apoptotic activity is believed to be due tothe agonistic activities of these two antibodies by themselves, as shownin FIG. 10. Accordingly, it is believed that the 3F11.39.7 antibody is ablocking Apo-2 antibody or an antibody which binds Apo-2 in a mode whichcompetes with binding of Apo-2 ligand to Apo-2.

Example 12

ELISA Assay to Test Binding of Apo-2 Antibodies to Other Apo-2 LigandReceptors

An ELISA was conducted to determine if the monoclonal antibody describedin Example 9 was able to bind other known Apo-2L receptors beside Apo-2.Specifically, the 3F11.39.7 antibody was tested for binding to DR4 [Panet al., supra], DcR1 [Sheridan et al., supra], and DcR2 [Marsters etal., Curr. Biol., 7:1003-1006 (1997)]. The ELISA was performedessentially as described in Example 9 above.

The results are shown in FIG. 11. The Apo-2 antibody 3F11.39.7 bound toApo-2. The 3F11.39.7 antibody also showed some cross-reactivity to DR4,but not to DcR1 or DcR2.

Example 13

Antibody Isotyping

The isotype of the 3F11.39.7 antibody (as described above) wasdetermined by coating microtiter plates with isotype specific goatanti-mouse Ig (Fisher Biotech, Pittsburgh, Pa.) overnight at 4° C. Theplates were then washed with wash buffer (as described in Example 9above). The wells in the microtiter plates were then blocked with 200 μlof 2% bovine serum albumin (BSA) and incubated at room temperature forone hour. The plates were washed again three times with wash buffer.Next, 100 μl of 5 μg/ml of purified 3F11.39.7 antibody was added todesignated wells. The plates were incubated at room temperature for 30minutes and then 50 μl HRP-conjugated goat anti-mouse IgG (as describedabove) was added to each well. The plates were incubated for 30 minutesat room temperature. The level of HRP bound to the plate was detectedusing HRP substrate as described above.

The isotyping analysis showed that the 3F11.39.7 antibody is an IgG1antibody.

Example 14 Single-Chain Apo-2 Antibodies

A. Antibody Phage Selection Using Streptavidin-Coated Paramagnetic Beads

A phage library was selected using soluble biotinylated antigen andstreptavidin-coated paramagnetic beads. The antigen, an Apo-2 ECDimmunoadhesin prepared as described in Example 2B above, wasbiotinylated using IMMUNOPURE NHS-biotin (biotiny-N-hydroxy-succinimide,Pierce) according to manufacturer's instructions.

Two panning experiments were performed. The first experiment wasdesigned to isolate phage clones specific for Apo-2 and which did notcross react with DR4 or DcR1. Three rounds of panning were carried out.For the first round, 10 μl of the Cambridge Antibody Technologies phagelibrary were blocked with 1 ml of MPBST (3% dry milk powder, 1×PBS, 0.2%TWEEN) containing 800 μg of CD4-Ig, 300 μg DR4-Ig, and 200 μg of DcR1-Igfor 1 hour on a rotating wheel at room temperature (CD4-Ig, DR4, andDcR1 are described in Capon et al., Nature, 337:525 (1989); Pan et al.,supra; and Sheridan et al., supra). Biotinylated Apo-2 ECD immunoadhesinwas then added to a final concentration of 100 nM, and phage wereallowed to bind antigen for 1 hour at 37° C. Meanwhile, 300 μl ofDYNABEADS M-280, coated with streptavidin (DYNAL) were washed 3 timeswith 1 ml MPBST (using a DYNAL Magnetic Particle Concentrator) and thenblocked for 2 hours at 37° C. with 1 ml fresh MPBST on a rotator. Thebeads were collected with the MPC, resuspended in 50 μl of MPBST, andadded to the phage-plus-antigen solution. Mixing continued on a wheel atroom temperature for 15 minutes. The DYNABEADS and attached phage werethen washed a total of 7 times: 3 times with 1 ml PBS-TWEEN, once withMPBS, followed by 3 times with PBS.

Phage were eluted from the beads by incubating 5 minutes at roomtemperature with 300 μl of 100 mM triethylamine. The phage-containingsupernatant was removed and neutralized with 150 μl of 1 M Tris-HCl (pH7.4). Neutralized phage were used to infect mid-log TG1 host cells andplated on 2YT agar supplemented with 2% glucose and 100 μg/mlcarbenicillin. After overnight growth at 30° C., colonies were scrapedinto 10 ml 2YT. 50 μl of this solution was used to inoculate 25 ml of2YT with carbenicillin and glucose and incubated, shaking, for 2 hoursat 37° C. Helper phage M13KO7 (Pharmacia) were added at a m.o.i. of 10.After adsorption, the cells were pelleted and resuspended in 25 ml of2YT with carbenicillin (100 μg/ml) and kanamycin (50 μg/ml) and growthcontinued at 30° C. for 4 hours. E. coli were removed from the phage bycentrifugation, and 1 ml of these phage (approximately 10¹² c.f.u.) wereused in subsequent rounds of selection.

For the second round of selection, the 1 ml of harvested phage wasadjusted to 3% dry milk, IX PBS, 0.2% TWEEN and then 100 μg DR4-Ig, 65μg DcR1-Ig, and 500 μg of CD4-Ig were added for blocking. For selection,biotinylated Apo-2 was added at 10 nM. Washing stringency was increasedto two cycles of 7 washes.

For the third round of selection, phage were blocked with only MPBST.Biotinylated Apo-2 was added to 1 nM, and washing stringency wasincreased to three cycles of 7 washes. Relatively few clones wereobtained in this round; therefore Pan 2B, Round 3 was performed using 5nM of biotinylated Apo-2 with all other conditions repeated as before.

A second panning experiment was performed similarly as above except thatin Rounds 1 and 2, blocking of phage solutions was conducted with MPBSTcontaining 1.0 mg/ml CD4-Ig (no other immunoadhesins) and Round 3 wasblocked with MPBST only. Biotinylated Apo-2 was added at 200 nM in Round1, 60 nM in Round 2, and 12 nM in Round 3. At each round, phage wereeluted from the magnetic beads with 300 μl of 100 nM triethylamine, thenwith 300 μl 0.1 M Tris-HCl (pH 7.5), and then with 300 μl glycine-0.1 MHCl (pH 2.2) containing 1 mg/ml BSA. The phage obtained from the threesequential elutions were pooled and used to infect host strain TG1 asabove.

B. ELISA Screening of Selected Clones

After each round of selection, individual carbenicillin-resistantcolonies were screened by ELISA to identify those producingApo-2-binding phage. Only those clones which were positive in two ormore assay formats were further studied.

Individual clones were inoculated into 2TY with 2% glucose and 100 μg/mlcarbenicillin in 96-well tissue culture plates and grown until turbid.Cultures were then infected at a m.o.i. of 10 with M12KO7 helper phage,and infected cells were transferred to 2YT media containingcarbenicillin (100 μg/ml) and kanamycin (50 μg/ml) for growth overnightat 30° C. with gentle shaking.

NUNC MAXISORP microtiter plates were coated with 50 μl per well of Apo-2ECD immunoadhesin, or CD4-IgG, at 2 μg/ml in 50 mM carbonate buffer (pH9.6), at 4° C. overnight. After removing antigen, plates were blockedwith 3% dry milk in PBS (MPBS) for 2 hours at room temperature.

Phage cultures were centrifuged and 100 μl of phage-containingsupernatants were blocked with 20 μl of 6×PBS/18% dry milk for 1 hour atroom temperature. Block was removed from titer plates and blocked phageadded and allowed to bind for 1 hour at room temperature. After washing,phage were detected with a 1:5000 dilution of horseradishperoxidase-conjugated anti-M13 antibody (Pharmacia) in MPBS followed by3′,3′,5′,5′-tetramethylbenzidine (TMB). Reactions were stopped by theaddition of H₂SO₄ and readings taken by subtracting the A_(405 nm) fromthe A_(450 nm).

C. DNA Fingerprinting of Clones

The diversity of Apo-2-binding clones was determined by PCR amplifyingthe scFv insert using primers pUC19R (5′AGC GGA TAA CAA TTT CAC ACA GG3′) (SEQ. ID. NO:12) which anneals upstream of the leader sequence andfdtetseq (5′GTC GTC TTT CCA GAC GGT AGT 3′) (SEQ. ID. NO:13) whichanneals in the 5′ end of gene III, followed by digestion with thefrequent-cutting restriction enzyme BstNI.

DNA Fingerprinting: Protocol

Mix A: dH2O 67 μl 10 x ampliTaq buffer 10 25 mM MgCl₂ 10 DMSO, 50% 2forward primer 1 Mix B: 2.5 mM dNTPs 8 μl AMPLITAQ 0.5 reverse primer1.090 μl of Mix A was placed in a reaction tube and then inoculated with avery small portion of E. coli colony using a yellow tip. The reactionmix was then heated in a PCR block to 98° C., for 3 minutes, removed,and placed on ice. 10 μl Mix B was then added and the reaction mix wasthermocycled at 95° C., 30 sec, 55° C. 30 sec, 72° C. 1 minute 20 sec,for 25 cycles in a Perkin Elmer 2400 thermocycler. 10 μl of theresultant reaction product was then removed and run on a 1% agarose gelto test for a 1 kB band. The remaining mix was brought to 1×BstNIreaction buffer, 5 units BstNI was added and the DNA was allowed todigest for 2 hours at 60° C. The resultant samples were thenelectrophoresed on a GeneGel Excel 12.5% acrylamide gel (PharmaciaBiotech).

D. Sequencing of Clones

The nucleotide sequence of representative clones of each fingerprintpattern were obtained. Colonies were inoculated into 50 ml of LB mediumsupplemented with 2% glucose and 100 μg/ml carbenicillin, and grownovernight at 30° C. DNA was isolated using Qiagen Tip-100s and themanufacturer's protocol and cycle sequenced with fluorescent dideoxychain terminators (Applied Biosystems). Samples were run on an AppliedBiosystems 373A Automated DNA Sequencer and sequences analyzed using theprogram “Sequencher” (Gene Codes Corporation). The nucleotides sequencesof selected antibodies 16E2, 20E6 and 24C4 are shown in SEQ ID NO:6, SEQID NO:7, and SEQ ID NO:8, respectively, (in FIGS. 15A, 15B and 15Crespectively). The corresponding amino acid sequences of antibodies16E2, 20E6 and 24C4 are shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ IDNO:11, respectively (and in FIG. 16). In addition, FIG. 16 identifiesthe signal region, and heavy and light chain complementarity determiningregions (underlined) of these scFv molecules. The CDR regions shown inFIG. 16 were assigned according to the methods of Kabat et al.,“Sequences of Proteins of Immunological Interest,” NIH Publ. No.91-3242, 5^(th) Edition.

E. Purification of scFvs with (his)₆

For protein purification of soluble antibody, E. coli strain 33D3 wastransformed with phagemid DNA. Five ml of 2YT with carbenicillin andglucose was used to grow overnight cultures at 30° C. 2.5 ml of thesecultures were diluted into 250 ml of the same media and grown to anOD₆₀₀ of approximately 1.2. The cells were pelleted and resuspended in500 ml of 2YT containing IPTG (1 mM) and carbenicillin (100 μg/ml) toinduce expression and grown for a further 16 hours at 22° C. Cellpellets were harvested and frozen at −20° C.

The antibodies were purified by immobilized metal chelate affinitychromatography (IMAC). Frozen pellets were resuspended in 10 ml ofice-cold shockate buffer (25 mM TRIS-HCl, 1 mM EDTA, 500 mM NaCl, 20%sucrose, 1 mM PMSF) by shaking on ice for 1 hour. Imidazole was added to20 mM, and cell debris removed by centrifugation. The supernatants wereadjusted to 1 mM MgCl₂ and 50 mM phosphate buffer pH 7.5. Ni-NTA agaroseresin from Qiagen was used according to the manufacturer's instructions.The resin was equilibrated with 50 mM sodium phosphate buffer pH 7.5,500 mM NaCl, 20 mM imidazole, and the shockate added. Binding occurredin either a batch mode or on a gravity flow column. The resin was thenwashed twice with 10 bed volumes of equilibration buffer, and twice withbuffer containing imidazole increased to 50 mM. Elution of proteins waswith 50 mM phosphate buffer pH 7.5, 500 mM NaCl and 250 mM imidazole.Excess salt and imidazole was removed on a PD-10 column (Pharmacia), andproteins were concentrated using a Centricom 10 to a volume of about 1ml.

Concentration was estimated spectrophotometrically assuming an A280 nmof 1.0=0.6 mg/ml.

F. Assays to Determine Binding Specificity of Anti-Apo-2 scFvs

To evaluate the specificity of each of the scFv clones, ELISA assayswere performed to evaluate binding of 16E2, 20E6 and 24C4 to Apo-2ECD-Ig, DR4-Ig, DcR1-Ig, DcR2-Ig and CD4-Ig (described above and inExample 12).

In brief, NUNC ELISA plates were coated with 50 μl of a 1 μg/mlreceptor-Ig immunoadhesin molecule in 0.05 M sodium carbonate buffer, pH9.5, and allowed to incubate overnight at 4° C. Plates were then blockedwith 285 μl ELISA diluent (PBS supplemented with 0.5% BSA, 0.05% Tween20, pH 7.4) for at least one hour at room temperature. 50 μl of thescFvs were added to the plates in a 1:5 serial dilution and allowed toincubate for 1 hour at room temperature. After this 1 hour dilution, theplates were washed 6 times with PBS/0.05% Tween. After binding toantigen coated plates, soluble scFv was detected by adding 50 μl of 1μg/ml Mab 9E10 (an anti-c-myc antibody; ATCC CRL 1729) per well andallowing the plates to incubate for 1 hour at room temperature. Afterwashing the plates 6 times with PBS/0.05% Tween, 50 μl of a 1:5000dilution of horseradish peroxidase-conjugated anti-Murine IgG antibody(Cappel catalogue: 55569) in MPBS was added to the plates and allowed toincubate for 1 hour. An observable signal was generated by adding 50 μlof 3′,3′,5′,5′-tetramethylbenzidine (TMB) peroxidase substrate (KPLcatalogue #: 50-76-00). Reactions were stopped by the addition of H₂SO₄and readings taken by subtracting the A_(405 nm) from the A_(450 nm).

As illustrated in FIGS. 12A, 12B and 12C, the ELISA assays showed thateach of these antibodies exhibited a relatively high degree ofspecificity for Apo-2.

Additional assays utilizing transfected cells also showed thespecificity of 16E2 antibody for Apo-2. Specifically,immunohistochemistry experiments were performed to evaluate the bindingspecificity of the 16E2 antibody to Apo-2 and DR4-transfected CHO cells.CHO cells were transfected with vector alone or vector containing thegene for Apo-2 or DR4. The transfected cells were removed from cultureplates, pelleted, and washed twice with PBS. The pellets were thenresuspended in O.C.T. (Fisher), flash frozen in isopentain and LN₂, andlater sectioned using standard protocols. Staining of the sectionedcells was performed using a Vectastain Elite ABC kit. The sections wereincubated with either anti-Apo-2 antibody 16E2 or a negative controlsingle chain antibody. The secondary antibody employed was either abiotinylated anti-c-myc 9E10 antibody or anti-Penta His antibody(Qiagen) followed by biotinylated anti-mouse IgG.

This immunohistochemistry assay showed specific staining of theApo-2-transfected cells but not the DR4-transfected cells. The cellularstaining was predominantly cytoplasmic.

Example 15

Assay for Ability of His-Tagged scFvs to Agonistically Induce Apoptosis

A. Annexin V-Biotin/Streptavidin-[S-35] 96 Well Assays

Purified scFv antibodies (as described in Example 14 above) were testedfor ability to induce Apo-2 mediated apoptosis.

In brief, SK-MES-1 cells (human lung carcinoma cell line; ATCC HTB 58)or HCT 116 cells (human colon carcinoma cell line; ATCC CCL 247) (4×10⁴cells/well) were aliquoted into 96 well plates in assay medium (1:1mixture of phenol-red free Dulbecco modified Eagle medium and phenol-redfree Ham's F-12 nutrient mixture supplemented with 10% fetal bovineserum, 2 mM L-glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin)and allowed to attach overnight at 37° C. The media was then removed and0.1 ml of assay medium containing scFv at a final concentration of 50ug/ml (16E2 or 20E6) was added to the wells (serial dilutions of 1:2performed in the plates) and allowed to incubate for 1 hour at roomtemperature. Other single chain antibodies were used as negativecontrols: an anti-tissue factor scFv clone, 7D5, or a scFv referred toas 19B8. After the 1 hour incubation with scFv antibody, 0.1 ml of 10ug/ml anti-His (Qiagen, cat. No. 1007671) or anti-c-myc antibodies wereadded to the appropriate wells. Wells not receiving a crosslinkingantibody received media alone. The plates were then allowed to incubatefor 30 minutes at room temperature. After the 30 minutes incubation, 0.1ml of 10 ug/ml goat anti-mouse IgG (ICN cst. No. 67-029) was added tothe appropriate wells. Wells not receiving anti-IgG antibody receivedmedia alone. The plates were then placed in an incubator for 15 minutesto allow the pH to return to 7.0. For positive controls, a 2 ug/mlsolution of Apo-2 ligand (Apo-2L) (prepared as described in Example 11)in potassium phosphate buffer at pH 7.0 was added to the appropriatewells, with serial 2 fold dilutions carried out in the plate. Thenegative control wells received media alone. The cells were thenincubated overnight at 37° C. in the presence of 5% CO₂. 0.05 ml ofannexin V-biotin (1 ug/ml) in 2×Ca²⁺ binding buffer (NeXins B.V.) wasthen added to the wells and then allowed to mix on a shaker for 30minutes. 0.05 ml of strepavidin-[S-35] (final concentration of 2.5×10⁴cpm/well) (Amersham) in 2×Ca²⁺ binding buffer was then added to thewells and then allowed to mix on a shaker for 30 minutes. The plateswere then sealed and centrifuged for 4 minutes at 1500 rpm. To assessthe extent of apoptosis, the plates were then counted on a TrialuxMicrobeta Counter (Wallace) to obtain cpm values corresponding toAnnexin-V binding.

As shown in FIGS. 13C and 14B, the 16E2 and 20E6 antibodiesagonistically induced apoptosis in SK-MES-1 cells.

B. Crystal Violet Assays

In addition to the annexin V-biotin/streptavidin-[S-35] assay describedabove, scFv antibodies (as described in Example 14 above) were testedfor activity to induce Apo-2 mediated apoptosis via assays utilizingcrystal violet.

In brief, the SK-MES-1 cells were plated at 4×10⁴ cells/well in assaymedium (described in Section A above) and allowed to attach overnight at37° C. The medium was removed and 0.1 ml of assay medium containing scFv(as described in Section A above) at a final concentration of 50 μg/mlwas added to the appropriate wells (wells without scFv added receive amedia change). Selected wells received “pre-complexed” samples in which10 ug/ml scFv 16E2 was combined with 100 ug/ml anti-His antibody for 5hours at 4° C. with continuous mixing before addition to the plate. Theplates were allowed to incubate for 1 hour at room temperature.

The scFv medium was removed and 0.1 ml of 10 μg/ml anti-His (Qiagen,cat. no. 1007671) or anti-c-myc antibodies diluted in assay medium wasadded to the wells (wells without crosslinker receive a media change.)The plates were then allowed to incubate for 30 minutes at roomtemperature.

The medium was then removed and 0.1 ml of 10 μg/ml Goat anti-Mouse IgG(Fc Fragment specific-ICN cst. no. 67-029) diluted in assay medium wasadded to the appropriate wells (wells without anti-Fc receive a mediachange). The plates were then placed in the incubator for 15 minutes toallow the pH to return to 7.0.

Apo-2L (stock at 100 μg/ml in potassium phosphate buffer pH 7.0) wasdiluted to 2 μg/ml and 0.1 ml was added to the appropriate wells. Serialtwo-fold dilutions were carried down the plate. The plates were thenincubated overnight at 37° C.

All medium was removed from the wells and the plates were then floodedwith crystal violet solution. The plates were allowed to stain for 15minutes. The crystal violet was removed by flooding the plates withrunning tap water. The plates were then allowed to dry overnight.

The plates were read on an SLT plate reader at 540 nm and the dataanalyzed using an Excel macro and 4p-fit.

As shown in FIGS. 13A, 13B, 14A and 14B, the 16E2 and 20E6 antibodiesagonistically induced apoptosis in SK-MES-1 cells.

Example 16

Assay for Ability of gD-Tagged scFvs to Agonistically Induce Apoptosis

A purified gD-tagged form of 16E2 scFv was tested for ability to induceApo-2 mediated apoptosis in a crystal violet assay as described inExample 15 above.

A. Construction of scFv with gD Tag

The Sfi I to Not I fragment of the scFv form of 16E2 was subcloned intoa derivative of pAK19 (Carter et al., Methods: A Companion to Methods inEnzymology, 3:183-192 (1991)) containing the phoA promoter and stIIsignal sequence rather than the lacZ promoter and hybrid signal sequenceof the original library. For ease of purification, a DNA fragment codingfor 12 amino acids (met-ala-asp-pro-asn-arg-phe-arg-gly-lys-asp-leu SEQID NO:14) derived from herpes simplex virus type 1 glycoprotein D (Laskyet al., DNA, 3:23-29 (1984)) was synthesized and inserted at the 3′ endof the VL domain in place of the (his)₆ and c-myc epitope originallypresent in the Cambridge Antibody Technologies library clones.

B. Expression in E. coli

The plasmid containing the gene for scFv 16E2-gD was transformed into E.coli strain 33D3 for expression in shake flask cultures. 5 ml of 2YTwith carbenicillin and glucose was used to grow overnight cultures at30° C. 2.5 ml of these cultures were diluted into 250 ml of the samemedium and grown to an OD₆₀₀ of approximately 1.0. The cells werepelleted and resuspended in 500 ml of Modified AP-5 Minimal Mediacontaining carbenicillin (100 μg/ml) and grown for an additional 16hours at 30° C. The cells were then pelleted and frozen.

C. Purification of scFv with gD Tag

Frozen cell paste was resuspended at 1 gm/10 ml of shockate buffer (25mM Tris-HCl, 1 mM EDTA, 500 mM NaCl, 20% sucrose, 1 mM PMSF, pH 7.2) andgently agitated 4 hours on ice. The cell suspension was then processedthrough a Polytron microfluidizer (Brinkman). Cell debris was removed bycentrifugation at 10,000×g for 30 minutes. After filtration through a0.22 micron filter, the supernatant was loaded onto an affinity column(2.5×9.0 cm) consisting of an anti-gD antibody 5B6 (Paborsky et al.,Protein Engineering, 3:547-553 (1990)) coupled to CNBr Sepharose whichhad been equilibrated with PBS. The column was washed 18 hours with PBSuntil the absorbance of the column effluent was equivalent to baseline.All steps were done at 4° C. at a linear flow rate of 25 cm/hour.Elution was performed with 0.1 M acetic acid, 0.5 M NaCl, pH 2.9. Columnfractions were monitored by absorbance at 280 nm and peak fractionspooled, neutralized with 1.0 M Tris, pH 8.0, dialyzed against PBS andsterile filtered. The resultant protein preparations were analyzed bynon-reducing SDS-PAGE.

D. Crystal Violet Assay

The apoptosis assay was performed essentially as described in Example15(B) above except that samples were serially diluted 1:3 in the platesand the 16E2-gD tagged antibody was tested in addition to two otherpreparations of 16E2 scFv (referred to as Prep. A and Prep. B in FIG.14C). The results of the assay showing apoptosis induction in SK-MES-1cells by 16E2-gD antibody are illustrated in FIG. 14C.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va., USA(ATCC):

Material ATCC Dep. No. Deposit Date pRK5-Apo-2 209021 May 8, 19973F11.39.7 HB-12456 Jan. 13, 1998

This deposit was made wider the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit and at least five years after the mostrecent request received by the depository for the furnishing of a sampleof the deposit. The deposit will be made available by ATCC under theterms of the Budapest Treaty, and subject to an agreement betweenGenentech, Inc., and ATCC, which assures permanent and unrestrictedavailability of the progeny of the culture of the deposit to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 U.S.C. Section 122 and the Commissioner's rules pursuant thereto(including 37 CFR Section 1.14 with particular reference to 8860G 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An isolated Apo-2 agonist monoclonal antibody which (a) binds toApo-2 polypeptide consisting of amino acid residues 1 to 411 of SEQ IDNO:1 and (b) induces apoptosis in at least one type of mammalian cancercell.
 2. The isolated agonist antibody of claim 1, wherein said antibodyis a chimeric antibody.
 3. The isolated agonist antibody of claim 1,wherein said antibody is a humanized antibody.
 4. The isolated agonistantibody of claim 1, wherein said antibody is a human antibody.
 5. Theisolated agonist antibody of claim 1, wherein said antibody is an Fabfragment.
 6. The isolated agonist antibody of claim 1, wherein saidantibody is a scFv fragment.
 7. The isolated agonist antibody fragmentof claim 1, wherein said antibody is a F(ab′)₂ fragment.
 8. The isolatedagonist antibody of claim 1, wherein said antibody comprises asingle-chain antibody.
 9. The single-chain antibody of claim 8 which isselected from the group consisting of 16E2, 20E6, and 24C4.
 10. Theisolated agonist antibody of claim 1 wherein said antibody inducesapoptosis in at least one type of mammalian cancer cell in vivo.
 11. Theisolated agonist antibody of claim 1 wherein said antibody inducesapoptosis in at least one type of mammalian cancer cell in vitro. 12.The isolated agonist antibody of claim 1 wherein said antibody inducesapoptosis in SK-MES-1 lung carcinoma cells in vitro.
 13. A compositioncomprising the agonist antibody of claim 1, 2, 3, 4, 5, 6, 7, 8, 10, 11,or 12 and a pharmaceutically acceptable carrier.
 14. An isolated Apo-2agonist monoclonal antibody which (a) binds to a soluble extracellulardomain sequence of an Apo-2 polypeptide consisting of amino acids 54 to182 of SEQ ID NO:1 and (b) induces apoptosis in at least one type ofmammalian cancer cell.
 15. The isolated agonist antibody of claim 14,wherein said antibody is a chimeric antibody.
 16. The isolated agonistantibody of claim 14, wherein said antibody is a humanized antibody. 17.The isolated agonist antibody of claim 14, wherein said antibody is ahuman antibody.
 18. The isolated agonist antibody of claim 14, whereinsaid antibody comprises an Fab fragment.
 19. The isolated agonistantibody of claim 14, wherein said antibody comprises a scFv fragment.20. The isolated agonist antibody of claim 14, wherein said antibodycomprises a F(ab′)₂ fragment.
 21. The isolated agonist antibody of claim14, wherein said antibody comprises a single-chain antibody.
 22. Thesingle-chain antibody of claim 21 which is selected from the groupconsisting of 16E2, 20E6, and 24C4 antibody.
 23. The isolated agonistantibody of claim 14 wherein said antibody induces apoptosis in at leastone type of mammalian cancer cell in vivo.
 24. The isolated agonistantibody of claim 14 wherein said antibody induces apoptosis in at leastone type of mammalian cancer cell in vitro.
 25. The isolated agonistantibody of claim 14 wherein said antibody induces apoptosis in SK-MES-1lung carcinoma cells in vitro.
 26. A composition comprising the agonistantibody of claim 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, or 25 and apharmaceutically acceptable carrier.
 27. An isolated Apo-2 agonistmonoclonal antibody which (a) binds to a soluble extracellular domainsequence of an Apo-2 polypeptide consisting of amino acids 1 to 182 ofSEQ ID NO:1 and (b) induces apoptosis in at least one type of mammaliancancer cell.
 28. The isolated agonist antibody of claim 27, wherein saidantibody is a chimeric antibody.
 29. The isolated agonist antibody ofclaim 27, wherein said antibody is a humanized antibody.
 30. Theisolated agonist antibody of claim 27, wherein said antibody is a humanantibody.
 31. The isolated agonist antibody of claim 27, wherein saidantibody comprises an Fab fragment.
 32. The isolated agonist antibody ofclaim 27, wherein said antibody comprises a scFv fragment.
 33. Theisolated agonist antibody of claim 27, wherein said antibody comprises aF(ab′)₂ fragment.
 34. The isolated agonist antibody of claim 27, whereinsaid antibody comprises a single-chain antibody.
 35. The single-chainantibody of claim 34 which is selected from the group consisting of16E2, 20E6, and 24C4.
 36. The isolated agonist antibody of claim 27wherein said antibody induces apoptosis in at least one type ofmammalian cancer cell in vivo.
 37. The isolated agonist antibody ofclaim 27 wherein said antibody induces apoptosis in at least one type ofmammalian cancer cell in vitro.
 38. The isolated agonist antibody ofclaim 27 wherein said antibody induces apoptosis in SK-MES-1 lungcarcinoma cells in vitro.
 39. A composition comprising the agonistantibody of claim 27, 28, 29, 30, 31, 32, 33, 34, 36, 37, or 38 and apharmaceutically-acceptable carrier.
 40. An isolated Apo-2 agonistmonoclonal antibody which (a) binds to Apo-2 polypeptide encoded by thecDNA contained in ATCC Deposit No. 209021 and (b) induces apoptosis inat least one type of mammalian cancer cell.
 41. The isolated agonistantibody of claim 40, wherein said antibody is a chimeric antibody. 42.The isolated agonist antibody of claim 40, wherein said antibody is ahumanized antibody.
 43. The isolated agonist antibody of claim 40,wherein said antibody is a human antibody.
 44. The isolated agonistantibody of claim 40, wherein said antibody comprises an Fab fragment.45. The isolated agonist antibody of claim 40, wherein said antibodycomprises a scFv fragment.
 46. The isolated agonist antibody of claim40, wherein said antibody comprises a F(ab′)₂ fragment.
 47. The isolatedagonist antibody of claim 40, wherein said antibody comprises asingle-chain antibody.
 48. The single-chain antibody of claim 47 whichis selected from the group consisting of 16E2, 20E6, and 24C4.
 49. Theisolated agonist antibody of claim 40 wherein said antibody inducesapoptosis in at least one type of mammalian cancer cell in vivo.
 50. Theisolated agonist antibody of claim 40 wherein said antibody inducesapoptosis in at least one type of mammalian cancer cell in vitro. 51.The isolated agonist antibody of claim 40 wherein said antibody inducesapoptosis in SK-MES-1 lung carcinoma cells in vitro.
 52. A compositioncomprising the agonist antibody of claim 40, 41, 42, 43, 44, 45, 46, 47,49, 50, or 51 and a pharmaceutically-acceptable carrier.